stable formulations of anti-tigit antibodies alone and in combination with programmed death receptor

专利摘要:
the present invention relates to stable formulations of antibodies against t cell immunoreceptor with ig and itim (tigit) domains, optionally also containing a human anti-programmed death receptor 1 (pd-1) antibody or antigen binding fragment thereof . methods of treating various cancers and chronic infections with the formulations of the invention are also provided.
公开号:BR112019022698A2
申请号:R112019022698
申请日:2018-05-01
公开日:2020-05-19
发明作者:De Arnab；Nachu Narasimhan Chakravarthy
申请人:Merck Sharp & Dohme；
IPC主号:

专利说明:

FORMULATION OF THERAPEUTIC ANTIBODIES AND USE OF THE SAME
FIELD OF THE INVENTION
[001] The invention relates to therapeutic antibody formulations and their use in the treatment of various disorders. In one aspect, the invention relates to formulations comprising antibodies or antigen-binding fragments thereof that bind to T cell immunoreceptors with Ig and ITIM domains (TIGIT). In another aspect, such a formulation further comprises a human anti-programmed death receptor (PD-1) antibody or antigen-binding fragment thereof. Methods of treating various cancers and chronic infections with the formulations of the invention are also provided.
CROSS REFERENCE TO RELATED ORDERS
[002] This application claims the benefit of U.S.S.N 62 / 500.278, deposited on May 2, 2017, the contents of which are incorporated in this document in its entirety for reference.
REFERENCE TO LISTING OF SEQUENCES ELECTRONICALLY SUBMITTED
[003] The sequence listing for this application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with the file name 24453WOPCT-SEQTXT01MAY2018.TXT, created on May 1, 2018, and a size 227 Kb. This list of strings submitted through the EFS-Web is part of the specification and is incorporated in this document for reference in its entirety.
BACKGROUND OF THE INVENTION
[004] Antibody drugs for use in humans may differ slightly in the amino acid sequence of their constant domains, or in their structural sequences within the variable domains, but they typically differ most dramatically in the CDR sequences. Even antibodies
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2/145 that bind to the same protein, the same polypeptide or even potentially the same epitope can comprise entirely different CDR sequences. Therapeutic antibodies for use in humans can also be obtained from human germline antibody sequences or from non-human germline antibody sequences (eg, rodents), as in humanized antibodies, leading to even greater sequence diversity of potential CDRs. These sequence differences result in different solution stability and different response capabilities to solution parameters. In addition, small changes in amino acid disposition or changes in one or a few amino acid residues can result in dramatically different antibody stability and susceptibility to sequence specific degradation trajectories. As a consequence, it is currently not possible to predict the solution conditions necessary to optimize the stability of antibodies. Each antibody must be studied individually to determine the optimal solution formulation. Bhambhani et al. (2012) J. Pharm. Sci.101: 1120.
[005] Antibodies are also relatively high molecular weight proteins (~ 150,000 Da), for example, compared to other therapeutic proteins like hormones and cytokines. As a consequence, it is often necessary to dose with relatively high amounts of antibody drugs to achieve the desired molar concentrations of drug. In addition, it is often desirable to administer antibody drugs sub-automatically, as this allows for self-administration. Self-administration avoids the time and expense associated with visits to medical facilities for administration, for example, intravenously. Subeutaneous release is limited by the volume of solution that can be practically delivered to the injection site in a single injection, which is usually about
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3/145 from 1 to 1.5 mL. Subcutaneous self-administration is typically performed using a pre-filled syringe or auto-injector filled with a liquid drug solution formulation, rather than a lyophilized form, to avoid the need for the patient to resuspend the drug before injection. Antibody drugs must be stable during storage to ensure efficacy and consistent dosing, so it is critical that any chosen formulation supports desirable properties, such as high concentration, clarity and acceptable viscosity, and that it also maintains these properties and efficacy of the drug in an acceptably long shelf life under typical storage conditions.
[006] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immunomodulatory receptor expressed mainly in activated T cells and NK cells. TIGIT is also known as VSIG9; VSTM3; and WUCAM. Its structure shows an extracellular immunoglobulin domain, a type 1 transmembrane region and two ITIM motifs. TIGIT forms part of a co-stimulating network consisting of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells and ligands expressed in APCs (CD155 and CD112).
[007] An important feature in the TIGIT structure is the presence of an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail domain. As with PD-1 and TIGIT, the ITIM domain in the cytoplasmic region of TIGIT is expected to recruit tyrosine phosphatases, such as SHP1 and SHP-2, and the subsequent dephosphorylation of tyrosine residues in the motifs of tyrosine immunoreceptor (ITAM) activation in subunits of the T cell receptor (TCR). Consequently, the binding of TIGIT by CD155 and CD112 receptor ligands expressed by tumor cells or TAMS can contribute to the suppression of TCR signaling and T cell activation, which is essential for the assembly of effective antitumor immunity. Thus, a
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4/145 TIGIT-specific antagonist antibody could inhibit CD155 and CD112-induced suppression of T cell responses and improve antitumor immunity.
[008] There is a need for stable formulations of anti-TIGIT antibodies for pharmaceutical use, for example, to treat various cancers and infectious diseases, as well as stable formulations of anti-TIGIT antibodies co-formulated with human anti-PD-1 antibodies. Preferably, these formulations will exhibit a long shelf life, will be stable when stored and transported, and will preferably exhibit stability over months to years under typical conditions for storing drugs for self-administration, that is, at refrigerator temperature in a syringe, resulting in a long shelf life for the corresponding drug product.
SUMMARY OF THE INVENTION
[009] In one aspect, the invention includes a formulation of an anti-TIGIT antibody or antigen binding fragment thereof comprising (i) an anti-TIGIT antibody or antigen binding fragment thereof; (ii) a buffer, (iii) a non-reducing sugar; (iv) a non-ionic surfactant; and (v) an antioxidant. In one embodiment, the formulation further comprises an anti-PD-1 antibody, for example, pembrolizumab or nivolumab. In another embodiment, the formulation comprises a chelator.
[010] In an embodiment of the invention, the formulation comprises (i) about 10 mg / ml to about 200 mg / ml of an anti-TIGIT antibody, or antigen binding fragment thereof; (ii) about 5 mM to about 20 mM buffer; (iii) about 6% to about 8% by weight / volume (w / v) of non-reducing sugar; (iv) about 0.01% to about 0.10% (w / v) of non-ionic surfactant; and (v) about 1 mM to about 20 mM of antioxidant. In one embodiment, the formulation
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5/145 further comprises an anti-PD-1 antibody, for example, pembrolizumab or nivolumab. In another embodiment, the formulation further comprises a chelator. In one embodiment, the formulation has a pH between 4.5 to 6.5. In particular embodiments, the pH of the formulation is about pH 5.5 to about pH 6.2. In an additional embodiment, the pH of the formulation is about pH 5.6 to about pH 6.0. In another embodiment, the pH of the formulation is about 5.7. In another embodiment, the pH of the formulation is about 5.8. In another embodiment, the pH of the formulation is about 5.9. In another embodiment, the pH of the formulation is about 6.0. In another embodiment, the pH of the formulation is about 6.1. In another embodiment, the pH of the formulation is about 6.2.
[011] In a formulation embodiment, the buffer is L-histidine or sodium acetate buffer, the non-reducing sugar is sucrose, the non-ionic surfactant is polysorbate 80, and the antioxidant is methionine, or a pharmaceutically acceptable salt thereof. . In one embodiment, the antioxidant is L-methionine. In another embodiment, the antioxidant is a pharmaceutically acceptable salt of Lmethionine, such as, for example, HCI of methionine.
[012] In another embodiment, the formulation comprises (i) about 10 mg / ml to about 200 mg / ml of an anti-TIGIT antibody, or antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM L-histidine buffer or about 5 mM to about 20 mM sodium acetate buffer; (iii) about 6% to about 8% w / v sucrose; (iv) about 0.01% to about 0.10% (w / v) of polysorbate 80; and (v) about 1 mM to about 20 mM L-methionine. In another embodiment, the formulation further comprises an anti-PD-1 antibody, for example, pembrolizumab or nivolumab. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μΜ to about 50 μΜ. In one embodiment, the chelator is DTPA. In another modality, the
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6/145 chelator is EDTA. In one embodiment, the buffer is L-histidine buffer. In one embodiment, the formulation comprises about 8 mM to about 12 mM L-histidine buffer. In another embodiment, the formulation comprises about 5 mM to about 10 mM L-methionine. In an additional embodiment, the formulation comprises polysorbate 80 in a weight ratio of approximately 0.02% (w / v). In one embodiment, the anti-TIGIT formulation comprises sucrose in a weight ratio of about 7% (w / v). In any of these modalities, methionine is L-methionine.
[013] In formulation embodiments, the concentration of the antiTIGIT antibody or antigen-binding fragment thereof is from about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / ml, 75 mg / ml or 100 mg / ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 20 mg / ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 25 mg / ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 50 mg / ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 75 mg / ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 100 mg / ml.
[014] In one aspect, a formulation is provided which comprises about 20 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[015] In one aspect, a formulation is provided which comprises about
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7/145 of 25 mg / mL of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% in w / v polysorbate 80 and about 10 mM L-methionine.
[016] In one aspect, a formulation is provided which comprises about 50 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[017] In one aspect, a formulation is provided which comprises about 75 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[018] In one aspect, a formulation is provided which comprises about 100 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.
[019] In one aspect of any of the above formulations, the formulation has a pH of about 5.4 to about 6.2. In another aspect, the formulation has a pH of about 5.5 to 6.2. In another embodiment, the formulation has a pH of about 5.8 to 6.1. In another embodiment, the pH is about 5.8. In one embodiment, the pH is 5.9. In another embodiment, the pH is 6.0. In an additional embodiment, the pH is 6.1.
[020] In one aspect of any of the above formulations, the formulation comprises an anti-PD1 antibody or antigen-binding fragment thereof. In one embodiment, the anti-PD1 antibody is pembrolizumab. In another aspect, the anti-PD1 antibody is nivolumab.
[021] In another aspect, the formulation can also comprise a chelator. In one embodiment, the chelator is DTPA. In one mode, the
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8/145 chelator is EDTA. In one aspect, the chelator is present in an amount of about μΜ to about 50 μΜ. In one embodiment, the formulation comprises about μΜ of the chelator. In one embodiment, the formulation comprises about μΜ of the chelator. In one embodiment, the formulation comprises about μΜ of the chelator. In one embodiment, the formulation comprises about 20 μΜ of the chelator. In one embodiment, the formulation comprises about 25 μΜ of the chelator. In one embodiment, the formulation comprises about 30 μΜ of the chelator. In one embodiment, the formulation comprises about 35 μΜ of the chelator. In one embodiment, the formulation comprises about 40 μΜ of the chelator. In one embodiment, the formulation comprises about 45 μΜ of the chelator. In one embodiment, the formulation comprises about 50 μΜ of the chelator. In one embodiment, the chelating agent is DTPA, which is present in any of the amounts set out above. In another embodiment, the chelating agent is EDTA which is present in any of the amounts set out above.
[022] In one embodiment, the formulation is contained in a glass bottle. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen at least below -70 ° C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.
[023] In certain embodiments, the formulation is stable at refrigerated temperature (2 to 8 ° C) for at least 3 months, preferably 6 months, and more preferably 1 year, and even more preferably up to 2 years. In a formulation embodiment, after 12 months at 5 ° C, the% of the anti-TIGIT antibody monomer is> 90% as determined by size exclusion chromatography. In another formulation, after 12 months at 5 ° C, the%
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9/145 of the anti-TIGIT antibody monomer is> 95% as determined by size exclusion chromatography. In another formulation embodiment, after 12 months at 5 ° C, the% of the heavy and light chain of the anti-TIGIT antibody is> 90% as determined by reducing CE-SDS. In another formulation embodiment, after 12 months at 5 ° C, the% of the heavy and light chain of the anti-TIGIT antibody is> 95% as determined by reducing CE-SDS. In another formulation embodiment, after 12 months at 5 ° C, the% intact IgG of the anti-TIGIT antibody is> 90% as determined by non-reducing CE-SDS. In another formulation embodiment, after 12 months at 5 ° C, the% of intact IgG of the anti-TIGIT antibody is> 95% as determined by non-reducing CE-SDS.
[024] In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof which comprises three light chain CDRs and three heavy chain CDRs, wherein the chain CDRs light comprise CDRL1 of SEQ ID NO: 111 or variant thereof, CDRL2 of SEQ ID NO: 112 or variant thereof, CDRL3 of SEQ ID NO: 113 or variant thereof and heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108 or variant thereof, CDRH2 of SEQ ID NO: 154 or variant thereof, and CDHR3 of SEQ ID NO: 110 or variant thereof. In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154 and CDHR3 of SEQ ID NO : 110. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen-binding fragment thereof
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10/145 comprises a heavy chain variable region comprising SEQ ID NO: 148 or variant thereof and a light chain variable region comprising SEQ ID NO: 152 or variant thereof. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof which comprises a heavy chain variable region comprising SEQ ID NO: 148 and a light chain variable region comprising SEQ ID NO: 152 In one aspect, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a human IgGl heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 or variant thereof and a constant domain of human kappa light chain comprising the amino acid sequence of SEQ ID NO: 293 or variant thereof. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgGl heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 and a human kappa light chain constant domain that comprises the amino acid sequence of SEQ ID NO: 293. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 or variant thereof and a constant domain of human kappa light chain comprising the amino acid sequence of SEQ ID NO: 293 or variant thereof.
[025] In one aspect, the invention provides a co-formulation of a
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11/145 anti-TIGIT antibody, or antigen binding fragment thereof and an anti-human PD-1 antibody, or antigen binding fragment thereof, comprising (i) an anti-TIGIT antibody, or binding fragment the antigen thereof; (ii) an anti-human PD-1 antibody or antigen-binding fragment thereof, (ii) a buffer, (iii) a non-reducing sugar; (iv) a non-ionic surfactant; and (v) an antioxidant. In one embodiment, co-formulation further comprises a chelator. In one embodiment, the chelator is EDTA. In another embodiment, the chelator is DTPA. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 1: 2. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 1: 1. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 2: 1.
[026] In one embodiment of the invention, co-formulation comprises (i) about 1 mg / ml to about 200 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) about 1 mg / ml to about 200 mg / ml of an anti-human PD-1 antibody; (iii) about 5 mM to about 20 mM buffer; (iv) about 6% to about 8% by weight / volume (w / v) of non-reducing sugar; (v) about 0.01% to about 0.10% (w / v) of non-ionic surfactant; and (vi) about 1 mM to about 20 mM antioxidant. In one embodiment, co-formulation further comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μΜ to about 50 μΜ. In one embodiment, the chelator is DTPA. In another embodiment, the chelator is EDTA. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 1: 2. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 1: 1. In a co-formulation modality, the ratio between the anti-human PD-1 antibody and the anti-TIGIT antibody is 2: 1. In one embodiment, co-formulation has a pH
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12/145 between 4.5 to 6.5. In particular embodiments, the pH of the formulation is about pH 5.5 to about pH 6.2. In an additional embodiment, the pH of the formulation is about pH 5.8 to 6.0.
[027] In a co-formulation modality, the buffer is Lhistidine buffer or sodium acetate buffer, the non-reducing sugar is sucrose, the non-ionic surfactant is polysorbate 80, and the antioxidant is L-methionine. In another embodiment, the co-formulation comprises (i) about 1 mg / ml to about 100 mg / ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) about 1 mg / ml to about 100 mg / ml of an anti-human PD-1 antibody or antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine or about 5 mM to about 20 mM sodium acetate buffer; (iv) about 6% to about 8% w / v sucrose; (v) about 0.01% to about 0.10% (w / v) of polysorbate 80; and (vi) about 1 mM to about 20 mM L-methionine. In one embodiment, the co-formulation optionally comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μΜ to about 50 μΜ. In one embodiment, the chelator is DTPA. In another embodiment, the chelator is EDTA. In a co-formulation modality, the buffer is L-histidine buffer. In one embodiment, the co-formulation comprises about 8 mM to about 12 mM L-histidine buffer. In another embodiment, the co-formulation comprises about 5 mM to about 10 mM L-methionine. In an additional embodiment, the co-formulation comprises polysorbate 80 in a weight ratio of approximately 0.02% w / v. In one embodiment, the co-formulation comprises sucrose in a weight ratio of about 7% (w / v).
[028] In co-formulation modalities, the concentration of the antiTIGIT antibody or antigen-binding fragment thereof is from about 1 mg / mL to about 100 mg / mL. In co-formulation modalities, the concentration of
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13/145 anti-TIGIT antibody or antigen-binding fragment thereof is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 10 mg / mL. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 12.5 mg / mL. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 20 mg / mL. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 25 mg / mL. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 50 mg / mL. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 75 mg / ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about or 100 mg / ml.
[029] In some co-formulation modalities, the concentration of human anti-PD-1 antibody is from about 1 mg / ml to about 100 mg / ml. In a co-formulation embodiment, the concentration of the anti-human PD-1 antibody is from about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is 20 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is 25 mg / ml.
[030] In one embodiment, the co-formulation comprises about 20 mg / ml of the anti-PD1 antibody, about 20 mg / ml of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[031] In one embodiment, the co-formulation comprises about 25 mg / ml of the anti-PD1 antibody, about 25 mg / ml of the anti-TIGIT antibody, 10
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14/145 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[032] In one embodiment, the co-formulation comprises about 50 mg / ml of the anti-PD1 antibody, about 50 mg / ml of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / w v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[033] In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the chain CDRs light comprise CDRL1 of SEQ ID NO: 111 or variant thereof, CDRL2 of SEQ ID NO: 112 or variant thereof, CDRL3 of SEQ ID NO: 113 or variant thereof and heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108 or variant thereof, CDRH2 of SEQ ID NO: 154 or variant thereof, and CDHR3 of SEQ ID NO: 110 or variant thereof. In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154, and CDHR3 of SEQ ID NO: 110. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof which comprises a heavy chain variable region comprising SEQ ID NO: 148 or variant thereof and a variable chain region comprising SEQ ID NO: 152 or a variant thereof. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof which comprises a heavy chain variable region comprising
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15/145
SEQ ID NO: 148 and a light chain variable region comprising SEQ ID NO: 152. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 or variant thereof and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 or variant thereof. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgGl heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 and a human kappa light chain constant domain that comprises the amino acid sequence of SEQ ID NO: 293. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 or variant thereof and a constant domain of human kappa light chain comprising the amino acid sequence of SEQ ID NO: 293 or variant thereof. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293.
[034] In one aspect of any of the formulations described above, the human anti-PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1 or variant thereof, CDRL2 of SEQ ID NO: 2 or variant thereof, CDRL3 of SEQ ID NO: 3 or variant thereof and heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6 or
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16/145 variant thereof, CDRH2 of SEQ ID NO: 1 or variant thereof and CDHR3 of SEQ ID NO: 8 or variant thereof. In one aspect of any of the formulations described above, the human anti-PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7 and CDHR3 of SEQ ID NO: 8. In another aspect, the formulations comprise an anti-human PDl antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 or variant thereof and a heavy chain variable region comprising SEQ ID NO: 9 or a variant thereof. In another aspect, the formulations comprise an anti-human PDl antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9. In another aspect, the formulations comprise an anti-human PDl antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 and a heavy chain comprising SEQ ID NO: 10. another aspect, the formulations comprise an anti-human PDl antibody or antigen-binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 or variant thereof and a heavy chain comprising SEQ ID NO: 10 or variant of same. In one aspect of any of the formulations described above, the anti-human PD1 antibody or antigen-binding fragment thereof is pembrolizumab. In another aspect, the human antiPD-1 antibody or antigen-binding fragment thereof is nivolumab.
[035] In one aspect of any of the co-formulations described above,
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17/145 the formulation comprises (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154 and CDHR3 of SEQ ID NO: 110 and (ii ) an anti-human PD-1 antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7 and CDHR3 of SEQ ID NO: 8.
[036] In one aspect of any of the above co-formulations, the formulation comprises (i) an anti-TIGIT antibody or antigen binding fragment thereof that comprises a heavy chain variable region comprising SEQ ID NO: 148 and a region light chain variable comprising SEQ ID NO: 152 and (ii) an anti-human PDl antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a chain variable region weight comprising SEQ ID NO: 9.
[037] In another aspect of any of the above co-formulations, the formulation comprises (i) an anti-TIGIT antibody or antigen binding fragment thereof which comprises a heavy chain variable region comprising SEQ ID NO: 148 and which further comprises a human IgGl heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 and a light chain variable region comprising SEQ ID NO: 152 and further comprising a human kappa light chain constant domain which comprises the amino acid sequence of SEQ ID NO: 293 and (ii)
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18/145 an anti-human PDl antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 and a heavy chain comprising SEQ ID NO: 10.
[038] In another aspect of any of the above co-formulations, the formulation comprises (i) an anti-TIGIT antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising SEQ ID NO: 148 and which further comprises a human IgGl heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 and a light chain variable region comprising SEQ ID NO: 152 and further comprising a human kappa light chain constant domain which comprises the amino acid sequence of SEQ ID NO: 293 and (ii) an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 and a heavy chain comprising SEQ ID NO : 10.
[039] In an embodiment of any of the formulations described above, the formulation is contained in a glass bottle. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen at least below -70 ° C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.
[040] The present invention provides a method of treating chronic infection or cancer in a mammalian individual (e.g., a human) in need thereof which comprises: administering an effective amount of the anti-TIGIT formulation or the co-formulation presented herein invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[041] Figure 1 shows the pH stability of the formulations over 9
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19/145 months in various storage conditions.
[042] Figure 2 shows the polysorbate 80 concentration stability of the formulations over 9 months under various storage conditions.
[043] Figure 3 shows the potency by ELISA stability data for formulations over 9 months under various storage conditions.
[044] Figure 4 shows the monomer (%) by UPSEC stability data for formulations over 9 months under various storage conditions.
[045] Figure 5 shows the species (%) of high molecular weight (HMW) by UP-SEC stability data for formulations over 9 months under various storage conditions.
[046] Figure 6 shows the species (%) of low molecular weight (LMW) by UP-SEC stability data for formulations over 9 months under various storage conditions.
[047] Figure 7 shows the purity of heavy chain + light chain (%) by stability data of reducing CE-SDS for formulations over 9 months under various storage conditions.
[048] Figure 8 shows the purity of intact IgG (%) by non-reducing CE-SDS stability data for formulations over 9 months under various storage conditions.
DETAILED DESCRIPTION OF THE INVENTION
[049] In one aspect, the invention provides formulations that comprise anti-TIGIT antibodies and antigen-binding fragments thereof that comprise methionine. Co-formulations of an anti-TIGIT antibody or antigen binding fragment thereof and a human anti-PD-1 antibody or antigen binding fragment thereof comprising methionine are also provided. In each case, the formulation and co-formulation
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20/145 optionally comprise a chelating agent.
I. Definitions and Abbreviations
[050] As used throughout the specification and attached claims, the following abbreviations apply:
API active pharmaceutical ingredient
CDR complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering system, unless otherwise indicated
CHO Chinese hamster ovary
Cl confidence interval
DTPA diethylenetriaminopentacetic acid
EC50 concentration resulting in 50% effectiveness or binding
ELISA enzyme-linked immunosorbent assay
Formalin-fixed FFPE, embedded in paraffin
FR structure region
HRP horseradish peroxidase
HNSCC squamous cell carcinoma of the head and neck
IC50 concentration resulting in 50% inhibition
IgG immunoglobulin G
IHC immunohistochemistry or immunohistochemistry mAb monoclonal antibody
2- (N-morpholino) ethanesulfonic acid MES
NCBI National Center for Biotechnology Information
NSCLC non-small cell lung cancer
PCR polymerase chain reaction
PD-1 programmed death 1 (better known as programmed cell death receptor 1 and programmed death receptor 1)
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PD-L1 ligand 1 of programmed cell death 1
PD-L2 ligand 2 of programmed cell death 1
PS80 polysorbate 80
TNBC triple-negative breast cancer
Vh immunoglobulin heavy chain variable region
VK kappa light chain variable region of immunoglobulin
Vl variable region of immunoglobulin light chain v / v volume by volume
WFI water for injection w / v weight by volume
[051] In order to make the invention easier to understand, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used in the present invention have the meaning commonly understood by an individual versed in the technique to which this invention belongs.
[052] As used throughout the specification and the appended claims, the singular forms one, one and / or include the plural reference, unless the context clearly indicates otherwise.
[053] The reference to or indicates one or two possibilities, unless the context clearly dictates one of the indicated possibilities. In some cases, and / or was used to highlight both possibilities.
[054] Treating or treating a cancer as used in the present invention means administering a formulation of the invention to an individual who has an immune condition or cancerous condition, or diagnosed with a cancer or pathogenic infection (e.g., viral, bacterial, fungal) , to achieve at least one positive therapeutic effect, such as, for example,
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22/145 reduced cancer cells, reduced tumor size, reduced cancer cell infiltration rate in peripheral organs or reduced rate of metastasis or tumor growth. Treatment may include one or more of the following: inducing / increasing an anti-tumor immune response, stimulating an immune response to a pathogen, toxin and / or autoantigen, stimulating an immune response to a viral infection, decreasing the number of one or more tumor markers, inhibit the growth or survival of tumor cells, eliminate or reduce the size of one or more lesions or cancerous tumors, decrease the level of one or more tumor markers, improve, reduce the severity or duration of cancer, prolong the survival of a patient in relation to the expected survival in a similar untreated patient.
[055] Immune condition or immune disorder encompasses, for example, pathological inflammation, an inflammatory disorder and an autoimmune disorder or disease. Immune condition also refers to infections, persistent infections and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors and cancers that resist eradication by the immune system. Cancerous condition includes, for example, cancer, cancer cells, tumors, angiogenesis and precancerous conditions like dysplasia.
[056] The positive therapeutic effects on cancer must be measured in several ways (See, W. A. Weber, J. Nucl. Med. 50:15 to 105 (2009)). For example, in relation to inhibition of tumor growth, according to the NCI standards, a T / C = 42% is the minimum level of antitumor activity. A T / C <10% is considered a high level of antitumor activity, with T / C (%) = average tumor volume of the treated / average tumor volume x 100. In some modalities, the treatment achieved by administering an
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The formulation of the invention is any progression-free survival (PFS), disease-free survival (DFS) or general survival (OS). PFS, also referred to as Time to Tumor Progression, indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time that patients have had a complete or partial response, as well as the amount of time that patients experienced a stable disease. DFS refers to the length of time during and after treatment when the patient remains disease-free. OS refers to an extension of life expectancy compared to naive or untreated individuals or patients. Although a modality of the formulations, methods of treatment and uses of the present invention may not be effective in achieving a positive therapeutic effect in all patients, it must be done in a statistically significant number of individuals as determined by any statistical test known in the art. such as the Student's t test, the chi ² test, the U test according to Mann and Whitney, the Kruskal-Wallis test (Η test), the Jonckheere-Terpstra test and the Wilcoxon test.
[057] The term patient (alternatively called an individual or individual in the present invention) refers to a mammal (eg, rat, mouse, dog, cat, rabbit) capable of being treated with the formulations of the invention, most preferably a human. In some situations, the patient is an adult patient. In other modalities, the patient is a pediatric patient.
[058] The term antibody refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, humanized antibodies, fully human antibodies and chimeric antibodies.
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Parental antibodies are antibodies obtained by exposing an immune system to an antigen before modifying the antibodies for their intended use, such as the humanization of an antibody for use as a human therapeutic antibody.
[059] In general, the basic structural unit of antibodies comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having a light chain (about 25 kDa) and a heavy chain (about 50 to 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. The carboxy-terminal portion of the heavy chain can define a constant region primarily responsible for the effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as mu, delta, gamma, alpha or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 more amino acids. See generally, Fundamental Immunology Chapter 7 (Paul, W., ed., 2- ed. Raven Press, N.Y. (1989).
[060] Typically, the variable domains of both heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within regions of relatively conserved structure (FR). CDRs are typically aligned with structure regions, allowing attachment to an epitope
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25/145 specific. In general, from the N-terminal to the C-terminal, both variable domains of light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al .; National Institutes of Health, Bethesda, Md .; 5 ^ ed .; Publ. NIH No. 91 to 3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1 to 75; Kabat, etal., (1977) J. Biol. Chem. 252: 6609 to 6616; Chothia, etal., (1987) J Mol. Biol. 196: 901 to 917 or Chothia, et al., (1989) Nature 342: 878 to 883.
[061] An antibody that specifically binds to a specified target protein is an antibody that exhibits preferential binding to that target compared to other proteins, but that specificity does not require absolute binding specificity. An antibody is considered specific for the intended target if its binding determines the presence of the target protein in a sample, for example, without producing unwanted results such as false positives. The antibodies or binding fragments thereof useful in the present invention will bind to the target protein with an affinity that is at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for non-target proteins. As used in the present invention, an antibody is said to specifically bind to a polypeptide that comprises a given amino acid sequence, for example, the amino acid sequence of a mature human TIGIT or human PD-1, if it binds to polypeptides that they understand that sequence, but they don't bind to proteins that don't have that sequence.
[062] Chimeric antibody refers to an antibody in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in an antibody derived from a particular species (eg
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26/145 example, human) or belonging to a particular antibody class or subclass, while the rest of the chain (or chains) is identical or homologous to corresponding sequences in an antibody derived from another species (for example, mouse) or belonging to another class or subclass of antibody, as well as fragments of those antibodies, as long as they exhibit the desired biological activity.
[063] Co-formulated or co-formulated as used in the present invention refers to at least two different antibodies or antigen-binding fragments thereof that are formulated together and stored as a combined product in a single vial or container (for example, a injection), instead of being formulated and stored individually and then mixed before administration or administered separately. In one embodiment, the co-formulation contains two different antibodies or antigen-binding fragments thereof.
[064] The term pharmaceutically effective amount or effective amount means an amount by which a sufficient therapeutic composition or formulation is introduced to a patient to treat the disease or condition. One skilled in the art recognizes that this level may vary according to the patient's characteristics such as age, weight, etc.
[065] The term about, when modifying the quantity (for example, mM, or M) of a substance or composition, the percentage (v / v or w / v) of a component of the formulation, the pH of a solution / formulation or the value of a parameter that characterizes a step in a method or similar refers to a variation in the numerical quantity that can occur, for example, through the typical measurement, handling and sampling procedures involved in the preparation, characterization and / or use the substance or composition; through instrumental error in these procedures; through differences in manufacturing,
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27/145 source or purity of the ingredients used to make or use the compositions or perform the procedures; and the like. In certain modalities, about can mean a variation of ± 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10%.
[066] As used in the present invention, x% (w / v) is equivalent to x g / 100 ml (for example, 5% w / v equals 50 mg / ml).
[067] The formulations of the present invention include antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.
[068] The terms cancer, cancerous or malignant refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (tract) cancer, kidney cancer, cancer of ovary, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, cancer stomach, bladder cancer, hepatoma, breast cancer, colon carcinoma and head and neck cancer.
[069] Chothia means an antibody numbering system described in Al-Lazikani et al., JMB 273: 927 to 948 (1997).
[070] Kabat, as used in the present invention, means an immunoglobulin numbering and alignment system, pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, Md.).
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[071] A growth inhibitory agent, when used in the present invention, refers to a compound or composition that inhibits the growth of a cell, especially the cancer cell that overexpresses any of the genes identified in the present invention, in vitro or in vivo . Thus, the growth inhibitory agent is one that significantly reduces the percentage of cells that express these genes in the S phase. Examples of growth inhibitory agents include agents that block the progression of the cell cycle (in a different location than the S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vincans (vincristine and vinblastine), taxanes and top II inhibitors such as doxorubicin, epirubicin, daunorubicin and etoposide. Agents that pause G1 also spread to stop in the S phase, for example, DNA alkylating agents, such as dacarbazine, mecloretamine and cisplatin. Additional information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled Cell cycle regulation, oncogens, and antineoplastic drugs by Murakami et al. (WB Saunders: Philadelphia, 1995).
[072] The terms TIGIT binding fragment, antigen binding fragment, binding fragment or fragment thereof encompass a fragment or an antibody derivative that still substantially retains its biological antigen binding activity (TIGIT and inhibits its activity (for example, blocking the binding of human TIGIT to its native ligands). Therefore, the term antibody fragment or TIGIT-binding fragment refers to a portion of a full-length antibody, generally binding to the antigen or variable region thereof. Examples of TIGIT antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments. Typically, a binding fragment or derivative retains at least 10% of its TIGIT inhibitory activity. In some modalities, a
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29/145 binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its TIGIT inhibitory activity, although any fragment binding with sufficient affinity to exert the desired biological effect is useful. In some embodiments, an antigen-binding fragment binds to its antigen with an affinity at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and more preferably at least 100 times greater than affinity with unrelated antigens. In one embodiment, the antibody has an affinity greater than about 10 ⁹ liters / mol, as determined, for example, by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107: 220 to 239. A TIGIT-binding fragment is also intended to include variants with conservative amino acid substitutions that do not substantially alter its biological activity.
[073] The terms PD-1 binding fragment, antigen binding fragment, binding fragment or fragment thereof encompass a fragment or an antibody derivative that still substantially retains its antigen-binding biological activity (Human PD-1) and inhibits its activity (for example, blocking the binding of PD-1 to PDL1 and PDL2). Therefore, the term antibody fragment or PD-1 binding fragment refers to a portion of a full-length antibody, generally binding to the antigen or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments. Typically, a binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, a binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its PD-inhibitory activity 1, although any binding fragment with sufficient affinity is useful to exert the biological effect
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Desired 30/145. In some embodiments, an antigen-binding fragment binds to its antigen with an affinity at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and more preferably at least 100 times greater than affinity with unrelated antigens. In one embodiment, the antibody has an affinity greater than about 10 ⁹ liters / mol, as determined, for example, by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem 107: 220 to 239. It is also intended that a PD-1 binding fragment can include variants with conservative amino acid substitutions that do not substantially alter its biological activity.
[074] Human antibody refers to an antibody that comprises only human immunoglobulin protein sequences. A human antibody can contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, mouse antibody or mouse antibody refers to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.
[075] Humanized antibody refers to forms of antibodies that contain sequences of non-human antibodies (eg, murines), as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of the at least one and, typically, two variable domains, where all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FR regions are those of a sequence of human immunoglobulin. The humanized antibody optionally will also comprise at least a portion of a constant region of
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31/145 immunoglobulin (Fc), typically that of a human immunoglobulin. Humanized forms of rodent antibodies generally comprise the same CDR sequences as parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody or for other reasons.
[076] The antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Patent ^Nos 5,624,821; W02003 / 086310; W02005 / 120571; W02006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58: 640-656. This modification can be used to improve or suppress various immune system reactions, with possible beneficial effects on diagnosis and therapy. Changes in the Fc region include changes in amino acids (substitutions, deletions and insertions), glycosylation or deglycosylation and addition of multiple Fc. Changes in Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent doses, with the concomitant greater convenience and less use of material. See, Presta (2005) 7. Allergy Clin. Immunol. 116: 731 at 734-35.
[077] Fully human antibody refers to an antibody that comprises only human immunoglobulin protein sequences. A fully human antibody can contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, mouse antibody refers to an antibody that comprises only mouse immunoglobulin sequences. A fully human antibody can be generated in a human, in a transgenic animal with human immunoglobulin germline sequences, by displaying phages or other molecular biological methods.
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[078] Hypervariable region refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR (for example, residues 24 to 34 (CDRL1), 50 to 56 (CDRL2) and 89 to 97 (CDRL3) in the variable domain of the light chain and residues 31 to 35 (CDRH1), 50 to 65 (CDRH2) and 95 to 102 (CDRH3) in the variable domain of the heavy chain, as measured by the Kabat numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5 ^ Ed Public Health Service, National Institutes of Health, Bethesda, Md.) And / or those residues from a hypervariable loop (ie residues 26 to 32 (Ll), 50 to 52 (L2) and 91 to 96 (L3) in light chain variable domain and 26 to 32 (Hl), 53 to 55 (H2) and 96 to 101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol.Biol. 196: 901 to 917 As used in the present invention, the term structure residues or FR refers to variable domain residues other than the residues of the hypervariable region in the present invention defined as residues of CDR. CDR and FR residues are determined according to the standard Kabat sequence definition. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
[079] Conservatively modified variants or conservative substitution refers to amino acid substitutions known to the elements versed in this technique and can be done generally without altering the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made according to those set out in Table 1 as follows:
Table 1. Exemplifying Conservative Amino Acid Substitutions
Original waste Conservative substitution Wing (A) Gly; To be
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Arg (R) Lys; His Asn (N) Gin; His Asp (D) Glu; Asn Cys (C) To be; Allah Gin (Q) Asn Glu (E) Asp; Gin Gly (G) Allah His (H) Asn; Gin lie (1) Read; Go Leu (L) He; Go Lys (K) Arg; His Met (M) Read; He; Tyr Phe (F) Tyr; Met; Read Pro (P) Allah Being (S) Thr Thr (T) To be Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Go (V) He; Read
[080] In addition, elements versed in this technique recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. See, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224 (4th Edition).
[081] The phrase essentially consists of or variations as it consists essentially of or consisting essentially of, as used throughout the specification and claims, indicates the inclusion of any elements
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34/145 or groups of elements recited and the optional inclusion of other elements of a similar or different nature to those mentioned, which do not materially alter the basic or new properties of the specified dosage regime, method or composition. As a non-limiting example, a linker compound consisting essentially of a recited amino acid sequence can also include one or more amino acids, including substitutions of one or more amino acid residues, which do not materially affect the properties of the linker.
[082] Understanding or variations as they understand, understand or understand and are used throughout the specification and claims in an inclusive sense, that is, to specify the presence of the declared resources, but not to prevent the presence or addition of additional features that they can materially improve the operation or utility of any of the modalities of the invention, unless the context requires otherwise due to the expressed language or necessary implication.
[083] Isolated antibody and isolated antibody fragment refer to the purification situation and, in this context, it means that the named molecule is substantially free from other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as debris cell phones and growth media. Generally, the term isolated is not intended to refer to a complete absence of this material or to an absence of water, buffers or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compound as described in the present invention. .
[084] Monoclonal antibody or mAb or Mab, as used in the present invention, refers to a population of substantially homogeneous antibodies, that is, the antibody molecules that make up the population are
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35/145 identical in the amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies with different amino acid sequences in their variable domains, particularly their CDRs, which are generally specific for different epitopes. The monoclonal modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring production of the antibody by any specific method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or can be produced by recombinant DNA methods (see, for example, US Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624 to 628 and Marks et al. (1991) J. Mol. Biol. 222: 581 to 597, for example. See also Presta (2005j J. Allergy Clin. Immunol. 116: 731.
[085] Tumor, as it applies to an individual diagnosed or suspected of cancer, refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or fluid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (blood cancer) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).
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[086] The term tumor size refers to the total size of the tumor that can be measured as the length and width of a tumor. The size of the tumor can be determined by a variety of methods known in the art, such as, for example, measuring the dimensions of the tumor (or tumors) after removal of the individual, for example, using forceps or while on the body using imaging techniques , for example, bone scan, ultrasound, computed tomography or magnetic resonance imaging.
[087] Variable regions or V region, as used in the present invention, means the segment of IgG chains that is variable in sequence between different antibodies. It extends to the Kabat residue 109 on the light chain and 113 on the heavy chain.
[088] The term buffer encompasses agents that maintain the pH of the solution of the formulations of the invention in an acceptable range or, for lyophilized formulations of the invention, provide an acceptable solution pH before lyophilization.
[089] The terms freeze-dried, freeze-dried and freeze dried refer to a process by which the material to be dried is first frozen and then the frozen ice or solvent is removed by sublimation in a vacuum environment. An excipient can be included in pre-lyophilized formulations to increase the stability of the lyophilized product in storage.
[090] The term pharmaceutical formulation refers to preparations that are in the form of allowing the active ingredients to be effective and that do not contain additional components that are toxic to the individuals to whom the formulation would be administered. The term formulation and pharmaceutical formulation are used interchangeably.
[091] Pharmaceutically acceptable refers to excipients (vehicles,
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37/145 additives) and compositions that can reasonably be administered to an individual to provide an effective dose of the active ingredient employed and that are generally considered safe, for example, physiologically tolerable and typically do not produce an allergic or similar unpleasant reaction such as gastric disorders and the like, when administered to a human. In another embodiment, this term refers to molecular entities and compositions approved by a federal or state government regulatory agency or listed in the US Pharmacopoeia or another pharmacopoeia generally recognized for use in animals and, more particularly, in humans.
[092] A reconstituted formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent, so that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (for example, parenteral administration) and can optionally be suitable for subcutaneous administration.
[093] Reconstitution time is the time required to rehydrate a lyophilized formulation with a solution to a clarified solution without particles.
[094] A stable formulation is one in which the protein in it essentially retains its physical stability and / or chemical stability and / or biological activity in storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed at Peptide and Protein Drug Delivery, 247 to 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev.10: 29 to 90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, in one embodiment, a stable formulation is a formulation with no significant changes observed at a
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38/145 refrigerated temperature (2 to 8 ° C) for at least 12 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2 to 8 ° C) for at least 18 months. In another embodiment, the stable formulation is a formulation with no significant changes observed at room temperature (23 to 27 ° C) for at least 3 months. In another embodiment, the stable formulation is a formulation with no significant changes observed at room temperature (23 to 27 ° C) for at least 6 months. In another embodiment, the stable formulation is a formulation with no significant changes observed at room temperature (23 to 27 ° C) for at least 12 months. In another embodiment, the stable formulation is a formulation with no significant changes observed at room temperature (23 to 27 ° C) for at least 18 months. The stability criteria for an antibody formulation are as follows. Typically, no more than 10%, preferably 5%, of antibody monomer is degraded as measured by SEC-HPLC. Typically, the formulation is colorless or transparent to slightly opalescent by visual analysis. Typically, the concentration, pH and osmolarity of the formulation have no more than +/- 10% change. The power is typically within 60 to 140%, preferably 80 to 120% of the control or reference. Typically, no more than 10%, preferably 5% of the cut of the antibody, i.e.,% of low molecular weight species, is observed, as determined, for example, by HP-SEC. Typically, no more than 10% is observed, preferably no more than 5% of the antibody aggregation, i.e.,% of high molecular weight species, as determined, for example, by HP-SEC.
[095] An antibody retains its physical stability in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation and / or denaturation after visual examination of color and / or clarity, or as measured by
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39/145 UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. Changes in protein conformation can be evaluated by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.
[096] An antibody retains its chemical stability in a pharmaceutical formulation if it does not show significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of the protein include hydrolysis or clipping (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in conjunction with mass spectroscopy or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focus, peptide mapping, isoaspartic acid measurement) and isomerization (assessed by measuring isoaspartic acid content, peptide mapping, etc.).
[097] An antibody retains its biological activity in a pharmaceutical formulation, if the biological activity of the antibody at any given time is within a predetermined range of the biological activity displayed at the time the pharmaceutical formulation was prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay.
[098] The term isotonic means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of about 270 to 328 mOsm. The slightly hypotonic pressure is 250 to 269 and the pressure
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40/145 slightly hypertonic is 328 to 350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or freezing ice osmometer.
II. Formulations and Coformulations of the invention.
[099] In one aspect, the invention provides biological formulations comprising anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to human TIGIT as an active pharmaceutical ingredient. The inclusion of methionine in these formulations reduces the oxidation of methionine residues in the Fc region of the anti-TIGIT antibody and, in the example of an anti-TIGIT antibody comprising a CDRH3 of SEQ ID NO: 110, tryptophan. Such formulations may further comprise a chelator, such as DTPA, which can further reduce oxidation.
[0100] In one aspect, the invention also provides a co-formulation of an anti-TIGIT antibody with an anti-PD-1 antibody. The main degradation pathways of pembrolizumab included the oxidation of methionine 105 (Metl05) in the heavy chain CDR3 (for example, M105 of SEQ ID NO: 10) after peroxide stress and oxidation of methionine residues Metl05 and Fc when exposed to light. Pembrolizumab maintained its bioactivity in most stress conditions for the levels of degradation tested. However, a reduction in affinity for PD-1 was observed for samples stressed by peroxide by Surface Plasmon Resonance (SPR). An exposed methionine residue or a methionine residue on an antibody's CDR has the potential to impact the biological activity of the antibody by oxidation. The addition of methionine is able to reduce Metl05 oxidation in the CDR of the pembrolizumab heavy chain.
Anti-PD-1 Antibodies and Antigen-Binding Fragments
[0101] In one aspect, the invention provides stable biological formulations
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41/145 which comprises anti-TIGIT antibodies or antigen-binding fragments thereof, co-formulated with human anti-PD-1 antibodies or antigen-binding fragments thereof which specifically bind to human PD-1 (for example, a human being) human or humanized anti-PD-1 antibody) as an active pharmaceutical ingredient (API PD-1), as well as methods for using the formulations of the invention. Any anti-PD-1 antibody or antigen-binding fragment thereof can be used in the co-formulations and methods of the invention. In particular embodiments, API PD-1 is an anti-PD-1 antibody, which is selected from pembrolizumab and nivolumab. In specific embodiments, the anti-PD-1 antibody is pembrolizumab. In alternative modalities, the anti-PD-1 antibody is nivolumab. Table 2 provides amino acid sequences for exemplary human anti-PD-1 antibodies pembrolizumab and nivolumab. The alternative PD-1 antibodies and antigen-binding fragments that are useful in the formulations and methods of the invention are shown in Table 3.
[0102] In some embodiments, an anti-human PD-1 antibody or antigen-binding fragment thereof for use in the co-formulations of the invention comprises three CDRL1, CDRL2 and CDRL3 light chain CDRs and / or three CDRH1 heavy chain CDRs , CDRH2 and CDRH3.
[0103] In one embodiment of the invention, CDRL1 is SEQ ID NO: 1 or a variant of SEQ ID NO: 1, CDRL2 is SEQ ID NO: 2 or a variant of SEQ ID NO: 2 and CDRL3 is SEQ ID NO: 3 or a variant of SEQ ID NO: 3.
[0104] In one embodiment, CDRH1 is SEQ ID NO: 6 or a variant of SEQ ID NO: 6, CDRH2 is SEQ ID NO: 7 or a variant of SEQ ID NO: 7 and CDRH3 is SEQ ID NO: 8 or a variant of SEQ ID NO: 8.
[0105] In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and the three heavy chain CDRs are SEQ ID NO: 6,
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SEQ ID NO: 7 and SEQ ID NO: 8.
[0106] In an alternative embodiment of the invention, CDRL1 is SEQ ID NO: 11 or a variant of SEQ ID NO: 11, CDRL2 is SEQ ID NO: 12 or a variant of SEQ ID NO: 12 and CDRL3 is SEQ ID NO: 13 or a variant of SEQ ID NO: 13.
[0107] In one embodiment, CDRH1 is SEQ ID NO: 16 or a variant of SEQ ID NO: 16, CDRH2 is SEQ ID NO: 17 or a variant of SEQ ID NO: 17 and CDRH3 is SEQ ID NO: 18 or a variant of SEQ ID NO: 18.
[0108] In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and the three heavy chain CDRs are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
[0109] In an alternative embodiment, the three light chain CDRs are SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and the three heavy chain CDRs are SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
[0110] In a further embodiment of the invention, CDRL1 is SEQ ID NO: 21 or a variant of SEQ ID NO: 21, CDRL2 is SEQ ID NO: 22 or a variant of SEQ ID NO: 22 and CDRL3 is SEQ ID NO: 23 or a variant of SEQ ID NO: 23.
[0111] In yet another embodiment, CDRH1 is SEQ ID NO: 24 or a variant of SEQ ID NO: 24, CDRH2 is SEQ ID NO: 25 or a variant of SEQ ID NO: 25 and CDRH3 is SEQ ID NO: 26 or a variant of SEQ ID NO: 26.
[0112] In another embodiment, the three light chain CDRs are SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 and the three heavy chain CDRs are SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
[0113] Some anti-human PD-1 antibodies and antigen-binding fragments of the invention comprise a light chain variable region and a heavy chain variable region. In some embodiments, the variable region of the light chain comprises SEQ ID NO: 4 or a variant of SEQ ID NO: 4, and the variable region of the heavy chain comprises SEQ ID NO: 9 or a variant of
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SEQ ID NO: 9. In other embodiments, the variable region of the light chain comprises SEQ ID NO: 14 or a variant of SEQ ID NO: 14, and the variable region of the heavy chain comprises SEQ ID NO: 19 or a variant of SEQ ID NO: 19. In other embodiments, the variable region of the heavy chain comprises SEQ ID NO: 27 or a variant of SEQ ID NO: 27 and the variable region of the light chain comprises SEQ ID NO: 28 or a variant of SEQ ID NO: 28, SEQ ID NO: 29 or a variant of SEQ ID NO: 29 or SEQ ID NO: 30 or a variant of SEQ ID NO: 30. In such embodiments, a variable region of light chain variable region or heavy chain is identical to the reference sequence, except having one, two, three, four or five amino acid substitutions. In some modalities, the substitutions are in the region of the structure (that is, outside the CDRs). In some embodiments, one, two, three, four or five of the amino acid substitutions are conservative substitutions.
[0114] In one embodiment of the co-formulations of the invention, the human anti-PD-1 antibody or antigen binding fragment comprises a light chain variable region that comprises or consists of SEQ ID NO: 4 and a heavy chain variable region that comprises or consists of SEQ ID NO: 9. In an additional embodiment, the human anti-PD-1 antibody or antigen binding fragment comprises a variable region of the light chain comprising or consisting of SEQ ID NO: 14 and a variable region of the heavy chain comprising or consisting of SEQ ID NO: 19. In one embodiment of the formulations of the invention, the human anti-PD-1 antibody or antigen binding fragment comprises a variable region of the light chain comprising or consisting of SEQ ID NO: 28 and a variable region of the heavy chain comprising or consisting of SEQ ID NO: 27. In an additional embodiment, the human anti-PD-1 antibody or antigen binding fragment
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44/145 comprises a variable region of the light chain comprising or consisting of SEQ ID NO: 29 and a variable region of the heavy chain comprising or consisting of SEQ ID NO: 27. In another embodiment, the antibody or fragment binding to antigen comprises a variable region of the light chain comprising or consisting of SEQ ID NO: 30 and a variable region of the heavy chain comprising or consisting of SEQ ID NO: 27.
[0115] In another embodiment, the co-formulations of the invention comprise an anti-human PD-1 antibody or antigen binding protein that has a V1 domain and / or a Vh domain with at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology with one of the V1 or Vh domains described above and exhibits specific binding to PD-1. In another embodiment, the human anti-PD-1 antibody or antigen-binding protein of the co-formulations of the invention comprises Vlc Vh domains with up to 1, 2, 3, 4 or 5 or more amino acid substitutions and exhibits specific binding to PD -1.
[0116] In any of the above embodiments, API PD-1 can be a full-length anti-PD-1 antibody or an antigen-binding fragment that specifically binds to human PD-1. In certain embodiments, API PD-1 is a full-length anti-PD-1 antibody selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE. Preferably, the antibody is an IgG antibody. Any IgG isotype can be used, including IgGi, IgGz, IgGs and IgGzi. Different constant domains can be attached to the V1 and Vh regions provided in the present invention. For example, if a particular intended use of an antibody (or fragment) of the present invention requires altered effector functions, a heavy chain constant domain other than IgG1 can be used. Although IgGl antibodies provide long half-lives and effector functions, such as complement activation and antibody-dependent cell cytotoxicity, these
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45/145 activities may not be desirable for all uses of the antibody. In such cases, a lgG4 constant domain, for example, can be used.
[0117] In the embodiments of the invention, API PD-1 is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set out in SEQ ID NO: 5 and a heavy chain which comprises or consists of a sequence of amino acid residues, as set out in SEQ ID NO: 10. In alternative embodiments, API PD-1 is an anti-PD-1 antibody that comprises a light chain that comprises or consists of a sequence of amino acid residues as set out in SEQ ID NO: 15 and a heavy chain comprising or consisting of a sequence of amino acid residues, as set out in SEQ ID NO: 20. In other embodiments, API PD-1 is an anti- PD-1 comprising a light chain comprising or consisting of a sequence of amino acid residues as set out in SEQ ID NO: 32 and a heavy chain comprising or consisting of a sequence of amino acid residues as set out in SEQ ID NO: 31. In m Additional odities, API PD-1 is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set out in SEQ ID NO: 33 and a heavy chain comprising or consisting of a amino acid residue sequence, as set out in SEQ ID NO: 31. In still further embodiments, API PD-1 is an anti-PD-1 antibody that comprises a light chain that comprises or consists of a sequence of amino acid residues such as established in SEQ ID NO: 34 and a heavy chain comprising or consisting of a sequence of amino acid residues, as set out in SEQ ID NO: 31. In some co-formulations of the invention, API PD-1 is pembrolizumab or a pembrolizumab biosimilar . In some formulations of the invention, the
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API PD-1 is nivolumab or a biosimilar to nivolumab.
[0118] Typically, the amino acid sequence variants of the anti-PD-1 antibodies and antigen binding fragments of the invention and the anti-TIGIT antibodies and antigen binding fragments will have an amino acid sequence with at least 75% identity of amino acid sequence with the amino acid sequence of a reference antibody or antigen binding fragment (e.g., heavy chain, light chain, Vh, Vl or humanized sequence), more preferably at least 80%, more preferably at least 85 %, more preferably at least 90% and most preferably at least 95, 98 or 99%. The identity or homology with respect to a sequence is defined in the present invention as the percentage of amino acid residues in the candidate sequence that are identical to the anti-PD-1 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitution as part of the sequence identity. No N-terminal, C-terminal or internal extensions, deletions or insertions in the antibody sequence should be interpreted as affecting the identity or homology of the sequence.
[0119] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same in equivalent positions when the two sequences are ideally aligned. The sequence identity can be determined using a BLAST algorithm, in which the parameters of the algorithm are selected to give the greatest correspondence between the respective sequences over the entire length of the respective reference sequences. The following references refer to BLAST algorithms frequently used for sequence analysis: ALGORITHMS BLAST: Altschul, S.F., et al., (1990) J. Mol. Biol. 215: 403 to 410; Gish, W., et al.,
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47/145 (1993) Nature Genet.3: 266 to 272; Madden, T.L. etal., (1996) Meth. Enzymol 266: 131 to 141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25: 3389 to 3402; Zhang, J., et al., (1997) Genome Res. 7: 649 to 656; Wootton, J.C., et al., (1993) Comput. Chem. 17: 149 to 163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. 10:67 to 70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., etal., A model of evolutionary change in proteins. In Atlas of Protein Sequence and Structure, (1978) volume 5, suppl.3. M.O. Dayhoff (org.), Pages 345-352, Natl. Biomed. Found., Washington, DC; Schwartz, R.M., et al., Matrices for detecting distant relationships. In Atlas of Protein Sequence and Structure, (1978) volume 5, suppl.3. M.O. Dayhoff (ed.), Pages 353-358, Natl. Biomed. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol.219: 555-565; States, DJ. et al., (1991) Methods 3: 66-70; Henikoff, S. et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 58735877; Dembo, A. et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, S.F. Evaluating the statistical significance of multiple distinct local alignments. In Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pages 1-14, Plenum, New York.
[0120] Likewise, any light chain class can be used in the compositions and methods described in the present invention. Specifically, kappa, lambda or variants thereof are useful in the present compositions and methods.
Table 2. Exemplifying PD-1 Antibody Sequences
Antibody Feature Amino Acid Sequence SEQ ID NO Pembrolizumab light chain CDR1 RASKGVSTSGYSYLH 1
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Antibody Feature Amino Acid Sequence SEQ ID NO CDR2 LASYLES 2 CDR3 QHSRDLPLT 3 Variable Region EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK 4 Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISS LEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTH HQ LSSPVTKSFN RG ECHeavy Pembrolizumab Chain CDR1 NYYMY 6 CDR2 GINPSNGGTNFNEKFKN 7 CDR3 RDYRFMGFDY 8 Variable Region QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAY Μ E LKS LQF D DTAVYYCA RRDYRFDMGF D Y WG QGTTVTVSS 9 JailHeavy QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAY Μ LKS LQF and D D Y DTAVYYCA RRDYRFDMGF WG HQ TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSGK 10 Nivolumab Light Chain CDR1 RASQSVSSYLA 11 CDR2 DASNR AT 12 CDR3 QQSSNWPRT 13 Variable Region EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 14
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Antibody Feature Amino Acid Sequence SEQ ID NO Light Chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPE FAVYYCQQSSN D WP RTFG QGTKVE1KRTVAAPSVF1F P PS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 15 Nivolumab heavy chain CDR1 NSGMH 16 CDR2 VIWYDGSKRYYADSVKG 17 CDR3 NDDY 18 Variable Region QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS 19 JailHeavy QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSK NTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 20
Table 3. Additional PD-1 Antibodies and Binding Fragments
Antigens Useful in Coformulations, Methods and Uses of the Invention.
A. Antibodies and antigen-binding fragments that comprise hPD-1.08A light and heavy chain CDRs in WO2008 / 156712 CDRL1 SEQ ID NO: 21 CDRL2 SEQ ID NO: 22 CDRL3 SEQ ID NO: 23 CDRH1 SEQ ID NO: 24 CDRH2 SEQ ID NO: 25 CDRH3 SEQ ID NO: 26 C. Antibodies and antigen-binding fragments that comprise the mature hlO9A heavy chain variable region and one of the variable chain regions
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light mature K09A in WO 2008/156712 Heavy chain VR SEQ ID NO: 27 Light chain VR SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 D. Antibodies and antigen-binding fragments comprising the mature 409 heavy chain and one of the mature K09A light chains in WO 2008/156712 Heavy chain SEQ ID NO: 31 Light chain SEQ ID NO: 32 or SEQ ID NO: 33 or SEQ ID NO: 34
[0121] In some forms of co-formulation of the invention, API PD-1 (ie, anti-PD-1 antibody or antigen-binding fragment thereof) is present in a concentration of about 25 mg / mL at about 100 mg / ml. In alternative embodiments, the API is present in a concentration of about 10 mg / ml, about 25 mg / ml, about 50 mg / ml, about 75 mg / ml or about 100 mg / ml.
Anti-TIGIT Antibodies and Antigen-Binding Fragment
[0122] In one aspect, the invention provides biological formulations comprising anti-TIGIT antibodies or antigen-binding fragments thereof, which specifically bind to human TIGIT (for example, a human or humanized anti-TIGIT antibody) as a pharmaceutical ingredient (API TIGIT), as well as methods for using the formulations of the invention.
[0123] In another aspect, the invention also provides biological co-formulations comprising (i) anti-TIGIT antibody or antigen-binding fragment thereof, which specifically binds to human TIGIT (for example, a human or anti-TIGIT antibody) humanized) and (ii) an anti-human PD-1 antibody or antigen-binding fragment thereof, which specifically binds to human PD-1. Any anti-TIGIT antibody or antigen-binding fragment thereof can be used in the formulation, including coformulation and methods of the invention. Exemplary anti-TIGIT antibody sequences are shown below in Tables 4 and 5.
TABLE 4. Exemplifying anti-TIGIT antibodies
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description SEQ ID NO: SEQUENCE 14A6 H - CDR1 35 SDYWG 14A6 H - CDR2 36 FITYSGSTSYNPSLKS 14A6 H - CDR3 37 MPSFITLASLSTWEGYFDF 14A6 L-CDR1 38 KASQSIHKNLA 14A6 L - CDR2 39 YANSLQT 14A6 L-CDR3 40 QQYYSGWT 14A6 VHPARENTAL 41 EVQLQESG PG LVKPSQSLSLTCSVTGSSIA SDYWGWIRKFPGNKMEWMGFITYSGSTS YNPSLKSRISITRDTSKNQFFLQLHSVTTD DTATYSCARMPSFITLASLSTWEGYFDFW GPGTMVTVSS 14A6 VLPARENTAL 42 D1QMTQS PS LLSASVG D R VTLN CKASQSI HKNLAWYQQKLG EA P K FLIYYANSLQTG IPSRFSGSGSGTDFTLTISGLQPEDVATYF CQQYYSGWTFGGGTKVELK HU14A6VH. 1 43 EVQLQESG PG LVKPSETLSLTCTVSG GSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS HU14A6VH. Ia 44 EVQLQESG PG LVKPSETLSLTCTVSG GSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCA RMPSFITLASLSTWEGYFDFW GQGTMVTVSS HU14A6VH.lb 45 EVQLQESG PG LVKPSETLSLTCTVSG GSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY N PSLKSRITISRDTSKNQFSLKLSSVTAAD TAVYYCA RMPSFITLASLSTWEGYFDFW
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description SEQ ID NO: SEQUENCE GQGTMVTVSS HU14A6VH. lc 46 EVQLQESG PG LVKPSETLSLTCTVSGSSIS SDYWGWIRQPPGKGLEWMGFITYSGSTS YNPSLKSRITISRDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.ld 47 EVQLQESG PG LVKPSETLSLTCTVSG GSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLHSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.le 48 EVQLQESG PG LVKPSETLSLTCTVSG GSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRITISRDTSKNQFSLKLHSVTAAD TAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.lf 49 EVQLQESG PG LVKPSETLSLTCTVSGSSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRITISRDTSKNQFSLKLHSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS HU14A6VH.lg 50 EVQLQESG PG LVKPSETLSLTCTVSGSSIS SDYWGWIRQPPGKGLEWMGFITYSGSTS YNPSLKSRITISVDTSKNQFSLKLHSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hui4A6VH.2 51 EVQLQESG PG LVKPSETLSLTCAVSG YSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.2a 52 EVQLQESG PG LVKPSETLSLTCAVSG YSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS Hul4A6VH.2b 53 EVQLQESG PG LVKPSETLSLTCAVSG YSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY
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description SEQ ID NO: SEQUENCE N PSLKSRITISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS Hul4A6VH.2c 54 EVQLQESG PG LVKPSETLSLTCAVSGSSIS SDYWGWIRQPPGKGLEWMGFITYSGSTS YNPSLKSRITISRDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.2d 55 EVQLQESG PG LVKPSETLSLTCAVSG YSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLHSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.2e 56 EVQLQESG PG LVKPSETLSLTCAVSG YSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRITISRDTSKNQFSLKLHSVTAAD TAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6VH.2f 57 EVQLQESG PG LVKPSETLSLTCAVSGSSIS SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRITISRDTSKNQFSLKLHSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS Hul4A6VH.2e 58 EVQLQESG PG LVKPSETLSLTCAVSGSSIS SDYWGWIRQPPGKGLEWMGFITYSGSTS YNPSLKSRITISRDTSKNQFSLKLHSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS Hul4A6Vk. 1 59 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTKVEIK Hul4A6Vk. Ia 60 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTKVEIK Hul4A6Vk. lb 61 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQK P G K A P K F L1YYANSLQTG1
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description SEQ ID NO: SEQUENCE PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTKVEIK Hul4A6Vk.2 62 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKLLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTKVEIK Hul4A6Vk.2a 63 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKFLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTKVEIK Hul4A6Vk.2b 64 DIQMTQSPSSLSASVGDRVTITCKASQSIH KNLAWYQQK P G K V P K F L1YYANSLQTG1 PSRFSGSGSGTDFTLTISSLQPEDVATYYC QQYYSGWTFGGGTKVEIK 16AHA_tigit_l 4a6_humaniza doVHl LB155.14A6.G 2.A8 VH1 65 EVQLQESGPGLVKPSETLSLTCTVSGSSIA SDYWGWIRQP PG KG LEWIGFITYSGSTSY NPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSSAS 18AHA_tigit _14a6_hum anizado_ VH2 LB155.14A6.G 2.A8 VH2 66 EVQLQESG PG LVKPSETLSLTCTVSGSSIA SDYWGWIRQPPG KG LEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS 20AHA_tigit _14a6_hum anizado_ VH3 LB155.14A6.G 2.A8 VH3 67 EVQLQESG PG LVKPSETLSLTCTVSGSSI A SDYWGWIRKPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS 21AHA_tigit _14a6_hum anizado_ VH4LB155.14A6.G2.A8 VH4 68 EVQLQESG PG LVKPSETLSLTCTVSGSSI A SDYWGWIRQPPGKKLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS 19AHA_tigit 69 EVQLQESG PG LVKPSETLSLTCTVSGSSI A
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description SEQ ID NO: SEQUENCE _14a6_hum anized_ VH5 LB155.14A6.G 2.A8 VH5SDYWGWIRQPPGKGMEWIGFITYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS 22AHA_tigit _14a6_hum anizado_ VH6 LB155.14A6.G 2.A8 VH6 70 EVQLQESG PG LVKPSETLSLTCTVSGSSIA SDYWGWIRKPPGKKMEWIGFITYSGSTS YNPSLKSRVTISRDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS 23AHA_tigit 14a6 humanized VH7 LB155.14A6. G2. A8 VH7 71 EVQLQESG PG LVKPSETLSLTCTVSGSSI A SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTADD TAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS 24AHA_tigit _14a6_hum anizado_ VH8 LB155.14A6. G2. A8 VH8 72 EVQLQESG PG LVKPSETLSLTCTVSGSSI A SDYWGWIRKPPGKKMEWIGFITYSGSTS YNPSLKSRVTISVDTSKNQFSLKLSSVTA A DTAVYYCA RMPSFITLASLSTWEGYFDF WGQGTMVTVSS 25AHA_tigit _14a6_hum anizado_ VH9 LB155.14A6. G2. A8 VH9 73 EVQLQESG PG LVKPSETLSLTCSVTGSSIA SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISRDTSKNQFSLKLSSVTAAD TAVYYCARMPSFITLASLSTWEGYFDFW GQGTMVTVSS 26AHA_tigit _14a6_hum anized_ VH10 LB155.14A6.G 2.A8 VH10 74 EVQLQQSGAG LLKPSETLSLTCSVTGSSIA SDYWGWIRQPPGKGLEWIGFITYSGSTSY NPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARMPSFITLASLSTWEGYFDF WGQGTMVTVSS 27AHA_tigit _14a6_hum anizado_ VH11 75 EVQLQESG PG LVKPPGTLSLTCSVTGSSIA SDYWGWVRQPPGKGLEWIGFITYSGSTS YNPSLKSRVTISVDTSKNQFSLKLSSVTA A DTAVYYCA RMPSFITLASLSTWEGYFDF
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description SEQ ID NO: SEQUENCE LB155.14A6.G2.A8 VH11WGQGTMVTVSS 09AHA_tigit _14a6_hum anizado_ VL1 LB155.14A6.G 2.A8 VL1 76 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTKVEIK llAHA_tigit 14a6 humanized VL2 LB155.14A6.G 2.A8 VL2 77 DIQMTQSPSSLSASVGDRVTITCKASQSIH KNLAWYQQK P G K A P K F L1YYANSLQTG V PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYSGWTFGGGTKVEIK 12AHA_tigit _14a6_hum anizado_ VL3LB155.14A6.G2.A8 VL3 78 D1QMTQS PSS LSASVG D RVTITCKASQSIH KNLAWYQQKPGKAPKLLIYYANSLQTGV PSRFSGSGSGTDFTLTISSLQPEDFATYFC QQYYSGWTFGGGTKVEIK 13AHA_tigit _14a6_hum anizado_ VL4 LB155.14A6.G 2.A8 VL4 79 D1 QMTQS PSS LSASVG D RVTITCKASQSIH KNLAWYQQKPGKAPKFLIYYANSLQTGV PSRFSGSGSGTDFTLTISSLQPEDFATYFC QQYYSGWTFGGGTKVEIK 15AHA_tigit _14a6_hum anizado_ VL5 LB155.14A6.G 2.A8 VL5 80 D1 QMTQS PSS LSASVG D RVTITCKASQSIH KNLAWYQQKPG KAPKLLIYYANSLQTG1 PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYSGWTFGGGTKVEIK 28H5 H - CDR1 81 GYSITSDYAWN 28H5 H - CDR2 82 YISNSGSASYNPSLKS 28H5 H - CDR3 83 LIYYDYGGAMNF 28H5 L- CDR1 84 KASQGVSTTVA
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description SEQ ID NO: SEQUENCE 28H5 L- CDR2 85 SASYRYT 28H5 L- CDR3 86 QHYYSTPWT 28H5 VHPARENTAL 87 DVQLQESG PG LVKPSQSLSLTCTVTG YSI TSDYAWNWIRQFPGNKLEWMGYISNSGS ASYNPSLKSRISITRDTSKNQFFLQLNSVT TEDTATYYCATLIYYDYGGAMNFWGQG TSVTVSS 28H5 VLPARENTAL 88 DIVMTQSHKFMSTSVGDRVSITCKASQGVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQSEDLAVYYCQHYYSTPWTFGGGTKLEIK 14H6 L variant of CDR2 89 YASNLQT 14H6 L variant of CDR2 90 YASSLQT 14H6 L variant of CDR2 91 YASTLQT 14H6 L variant of CDR2 92 YATTLQT 14H6 L variant of CDR2 93 YASYLQT 14H6 L variant of CDR2 94 YANQLQT 14H6 L variant of CDR2 95 Y AG SLQT 14H6 L variant of CDR2 96 YASQLQT 14H6 L variant of 97 YADSLQT
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description SEQ ID NO: SEQUENCE CDR2 14H6 L variant of CDR3 98 QQYYSGFT 14H6 L variant of CDR3 99 QQYYSGYT 14H6 L variant of CDR3 100 QQYYSGIT 14H6 L variant of CDR3 101 QQYYSGVT 14H6 L variant of CDR3 102 QQYYSGLT 14H6 L variant of CDR3 103 MPSFITLASLSTFEGYFDF 14H6 L variant of CDR3 104 MPSFITLASLSTYEGYFDF 14H6 L variant of CDR3 105 MPSFITLASLSTIEGYFDF 14H6 L variant of CDR3 106 MPSFITLASLSTVEGYFDF 14H6 L variant of CDR3 107 MPSFITLASLSTLEGYFDF 31C6 H -CDR1 108 SYVMH 31C6 H -CDR2 109 YIDPYNDGAKYNEKFKG 31C6 H-CDR3 110 GGPYGWYFDV 31C6 L-CDR1 111 RASEHIYSYLS
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description SEQ ID NO: SEQUENCE 31C6 L - CDR2 112 NAKTLAE 31C6 L - CDR3 113 QHHFGSPLT 31C6 VHPARENTAL(with CDRsunderlined) 114 EVQLQQSGPELVKPGSSVKMSCKASGYT FSSYVMHWVKQKPGQGLEWIGYIDPYND GAKYNEKFKGKATLTS D KSSSTAY M E LS SLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSS 31C6 VLPARENTAL (with CDRsunderlined) 115 D1QMTQS P AS LSASVG ETVTITCRASEHIY SYLSWYQQKQG KSPQLLVYNAKTLAEG VPSRFSGSGSGTQFSLKINSLQPEDFGTYY CQHHFGSPLTFGAGTTLELK 31C6 H VARIANT TO CDR2 (D56R) 116 YIDPYNrGAKYNEKFG 31C6 H VARIANT TO CDR2 (D56L) 117 YIDPYN1GAKYNEKGF 31C6 H VARIANT TO CDR2 (D56K) 118 YIDPYNkGAKYNEKFG 31C6 H VARIANT TO CDR2 (D56F) 119 YIDPYNfGAKYNEKFG 31C6 H VARIANT TO CDR2 (D56S) 120 YIDPYNsGAKYNEKFG 31C6 H VARIANT TO CDR2 (D56Y) 121 YIDPYNyGAKYNEKFG 31C6 H VARIANT TO CDR2 (D56V) 122 YIDPYNvGAKYNEKFG 31C6 H VARIANT TO CDR2 (G57R) 123 YIDPYNDrAKYNEKFKG
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description SEQ ID NO: SEQUENCE 31C6 H VARIANT TO CDR2 (G57N) 124 YIDPYNDnAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57Q) 125 YIDPYNDqAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57E) 126 YIDPYNDeAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57L) 127 YIDPYNDIAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57K) 128 YIDPYNDkAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57S) 129 YIDPYNDsAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57Y) 130 YIDPYNDyAKYNEKFKG 31C6 H VARIANT TO CDR2 (G57V) 131 YIDPYNDvAKYNEKFKG 31C6 L variant of CDR2 (N50A) 132 AAKTLAE 31C6 L variant of CDR2 (N50Y) 133 YAKTLAE 31C6 L variant of CDR2 (N50W) 134 WAKTLAE 31C6 L variant of CDR2 (N50S) 135 SAKTLAE
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description SEQ ID NO: SEQUENCE 31C6 L variant of CDR2 (N5 OT) 136 TAKTLAE 31C6 L variant of CDR2 (N500) 137 IAKTLAE 31C6 L variant of CDR2 (N50V) 138 VAKTLAE 31C6 L variant of CDR2 (A51N) 139 NNKTLAE 31C6 L variant of CDR2 (A510) 140 NIKTLAE 31C6 L variant of CDR2 (A51L) 141 NLLTLAE 31C6 L variant of CDR2 (A51T) 142 NTKTLAE 31C6 L variant of CDR2 (A51V) 143 NVKTLAE 31C6HUMZV Hl (with underlined CDRs) 144 EVQLVQSGAEVKKPGASVKVSCKASGYT FSSYVMHWVRQAPGQRLEWIGYIDPYND G AKYSQKFQG RVTLTRDTSASTAYM AND LS SLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS 31C6HUMZV H2 (with underlined CDRs) 145 EVQLVQSGAEVKKPGASVKVSCKASGYT FSSYVMHWVRQAPGQRLEWIGYIDPYND G AKYSQKFQG RVT LTS D KS ASTAY M E LS SLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS 31C6HUMZV H3 (with underlined CDRs) 146 EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYND G AKYAQKFQG RVTLTRDTSTSTVYM AND LS SLRSEDTAVYYCARGGPYGWYFDVWGQ
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description SEQ ID NO: SEQUENCE GTTVTVSS 31C6_HUMZ_ VH4 (with underlined CDRs) 147 EVQLVQSGAEVKKPGASVKVSCKASGYT FSYVMHWVRQAPGQGLEWIGYIDPYND G AKYAQKFQG RVTLTSD KSTSTVYM AND LS SLRSEDTAVYYCARGGPYGWYFDVWGQ GTTVTVSSSS 31C6_HUMZ_ VH5 (with underlined CDRs) 148 EVQLVQSGAEVKKPGSSVKVSCKASGYT FSSYVMHWVRQAPGQGLEWIGYIDPYND G AKYAQKFQG RVTLTS D KSTSTAYM AND LS SLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS 31C6_HUMZ_VH6 (withUnderlined CDRs) 149 EVQLVQSGAEVKKPGASVKVSCKASGYT FSSYVMHWVRQAPGQGLEWIGYIDPYND GAKYAQKFQGRVTLTSDKSISTAYM AND LS R LRS D DTVVYYCARGGPYGWYFDVWG Q GTTVSSSS 31C6_Humz_ LI(with CDRsunderlined) 150 DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK 31C6_Humz_ L2(with CDRsunderlined) 151 DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK 31C6_Humz_L 3 (with underlined CDRs) 152 DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHFGSPLTFGQGTRLEIK 31C6_Humz_L 4 (with underlined CDRs) 153 DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAEGVPSRFSGSGSGTQFTLTIS SLQPEDVATYYC QHHFGSPLTFGQGTRLEIK 31C6 H variant of CDR2 154 YIDPYNDGAKYAQKFQG 31C6 H variant of CDR2 155 YIDPYNDGAKYSQKFQG
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description SEQ ID NO: SEQUENCE 18G10 -VH sequence 156 QVQLM ESG PG LVQPSQTLSLTCTVSG FPL TSYTVHWVRQPPGKGLEWIGAIWSSGST DYNSALKSRLNINRDSSKSQVFLKMNSLQ TEDTAIYFCTKSGWAFFDYWGQGVMVT VSS 18G10 -VL sequence 157 DIQMTQSPSLLSASVGDRVTLNCIASQNIY KSLAWYQLKLG EAP KLLIYNANSLQAG1P SRFSGSGSGTDFALTISGLQPEDVATYFCQ QYSGGYTFGAGTKLELK 11A11 -VH sequence 158 EVQLVESGG DLVQPG RSLKISCVASG FTF SDYYMAWVRLAPQKGLEWVASISYEGS RTHYGDSVRGRFIISRDNPKNILYLQMNS LGSEDTATYFCARHTGTLDWLVYWGQG TLVIVSS 11A11 -VL sequence 159 NIVMAQSPKSMSISAGDRVTMNCKASQNVDNNIAWYQQKPGQSPKLLIFYASNRYSGVPDRFTGGGYGTDFTLTIKSVQAEDAAFYYCQRIYNFPTFGSGTKLEIK 14A6 H - CDR3 CONSENSUS 160 MPSFITLASLSTXEGYFDFX = W, F, Y, 1, V, L 14A6 L-CDR2 CONSENSUS 161 YAX1X2LQT Xi = N, S, T, G, D X ₂ = S, N, S, T, Y, Q 14A6 L-CDR3 CONSENSUS 162 QQYYSGXTX = W, F, Y, 1, V, L 14A6 VHPARENTAL CONSENSUS 163 EVQLQX1SGX2G LX3KPX4X5X6LSLTCX7VX8GX30SIX31SDYWGWX9RX10X11PGX12X13X14EWX15GFITYSGSTSYNPSLKSRX16X17IX18X19DTSKNQFX20LX21LX22SVTX23X24DTAX25Y
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description SEQ ID NO: SEQUENCE X26CARMPSFITLASLSTX27EGYFDFWGX32 GTX28X29TVSS Xi = E or Q X ₂ = P or A X ₃ = V or L X4 = S or P Xs = Q or E or G Xê = S or T X = S or T or A Xs = T or S X ₉ = 1 or V Xio = K or Q Xn = F or P Xi2 = N or K Xi3 = K or G Xi4 = M or L Xi5 = M or 1 X16 = 1 or V X17 = S or T X18 = T or S Xi9 = R or V XzcF F or SX ₂ i = Qor K X ₂₂ = H or S X23 = T OR A X24 = D OR A X ₂ 5 = T or V X ₂ 6 = S or Y, X ₂ 7 = W, F, Y, 1, V or LX ₂ 8 = Μ, V, L, A, R, N, P, Q, E, G, 1, Η, K, F,
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description SEQ ID NO: SEQUENCE S, T, W or Y X29 = V, T or L X3o = S or G or Y Xsi ⁼ A or S X3i = P or Q 14A6 VHHUMANIZED CONSENSUS 164 EVQLQX1SGX2G LX3KPX4X5TLSLTCX6VX7 GX3SIX9SDYWGWX10RX11X12PG KX13X14E WX15GFITYSGSTSYNPSLKSRX16TISX17D TSKNQFSLKLX18SVTAX19DTAVYYCARM PSFITLASLSTX20EGYFDFWGQGTX21X22T VSS Xi = E or Q _X2 = Q or X ₃ = V or L X4 = S or P X ₅ = E or G Xg = T or A or S X ₇ = S or T Xs = G or I Y X9 = S or A Χιο = 1 or V Xn = Q or K Xi2 = P or F Xi3 = G or K Xi4 = L or M Xis = 1 or M X16 = V or 1 Xi7 = V or R Xis = S or H Xi9 = A or D
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description SEQ ID NO: SEQUENCE X20 = W, F, Y, 1, V, LX21 = Μ, V, L, A, R, N, P, Q, E, G, 1, Η, K, F,S, T. W or YX22 = V, T or L 14A6 VLPARENTAL CONSENSUS 165 D1 QMTQS PSXi LSASVG DR VTX2X3C KASQ SIHKNLA WYQQKX4GX5X15 P KX ₆ LIYYAX7 X8LQTGX9PSRFSGSGSGTDFTLTISX10LQP EDX11ATYX12CQQYYSGX13TFGGGTKVE X14K Xi = G or S X 2 = L or 1 X ₃ = C OUT _X4 = L or P X ₅ = E or K , X ₆ = F or L X ₇ = C , S, T, G or DX ₈ = S, N, T, You Q X ₉ = 1 or V X10 = G or S X11 = V or F X12 = F or Y Xi3 = W, F, Y, 1, V or L Xi4 = L or 1 X15 = A or V 14A6 VLHUMANIZED CONSENSUS 166 D1 QMTQS PSS LSASVG D RVTITCKASQSIH KNLAWYQQKPG KX ₆ PKXiLI YYAX2X3LQ tgx ₄ psrfsgsgsgtdftltisslqpedx ₇ a TYYCQQYYSGX5TFGGGTKVEIK Xi, or F XI, or
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description SEQ ID NO: SEQUENCE X3 = S, N, T, Y or Q X ₄ = V or 1 X ₅ = W, F, Y, 1, V or L X ₆ = A or V X ₇ = F or V 31C6 H -CDR2 CONSENSUS 167 Yl DPYNX1X2AKYX3X4KFX5G Xi = D, R, L, K, F, S, You V X ₂ = G, R, N, Q, E, LK, S. You V X ₃ = N, A or S X ₄ = E or Q X ₅ = K or Q 31C6 L— CDR2 CONSENSUS 168 X1X2KTLAE Xi = N, A, V, W, S, TR, HG, 1 or V X ₂ = A, N, 1, L, T or V 31C6 VHPARENTAL CONSENSUS 169 EVQLXiQSGX ₂ EX ₃ X ₄ KPGX5SVKX ₆ SCKAS GYTFSSYVMHWVX7QX8PGQX9LEWIGYI DPYN XioXiiAKYXi2Xi ₃ KFXi ₄ GXisXi6TLTXi ₇ DXi8SXi9STX2LYM22. ₃ DX2 ₄ X2sVYYCARGGPYGX26YFDVWGX2 ₇ GT TVTVSS Xi = Qor V X ₂ = P or A X ₃ = V or L X ₄ = V or K Xs = S or A X ₆ = M or V X ₇ = K or R X ₈ = K or A Xg = G OR R Xio = D, R, L, K, F, S, You V
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description SEQ ID NO: SEQUENCE Xn = G, R, N, Q, E, L K, S, Y or VXi2 = N, A or SXi3 = E or QXi4 = K or QXi5 = R or KXi6 = A or VXi7 = S or RX18 = K or TX19 = S, 1, A or TX20 = A or VX21 = R or SX22 = T or RX23 = D or EX24 = S or TX25 = A or VX26 = W, A, D, E, F, G, 1, K, N, Q, R, S, T, Vor YX27 = A or Q 31C6 HUMANIZED VH CONSENSUS 170 EVQLVQSGAEVKKPGXiSVKVSCKASGY TFSSYVMHWVRQAPGQX2LEWIG YIDPYNX3X4AKYX5X5KFX7GRVTLTX8DX9 SX10STX11YMELSX12LRSX13DT X14VYYCARGGPYGX15YFDVWGQGTTVT VSS Xi = A or S X2 = R or G , _X3 = D, R, L, K, F, S, You V _X4 = L, R, C, Q, E, KL, S, Y, or VX ₅ = N, A or S Xõ = E or Q X ₇ = K or Q
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description SEQ ID NO: SEQUENCE Xs = R or S X ₉ = T or K Xio = A, T or 1 Xn = A or V Xi2 = S or R Xi3 = E or D Xi4 = A or V Xis = W, A, D, E, F, G , 1, K, N, Q, R, S, TV or Y 31C6 VLPARENTAL CONSENSUS 171 DIQMTQSPX1SLSASVGX2X.3VTITCRASEH IYSYLSWYQQKX _4G KX5PX6LLX7YX8X9KT LAEGVPSRFSGSGSGTX10FX11LX12IX13SL QP and DXuXisTYYCQH H FGSPLTFGXigGTXu LEXisK Xi = A or S X ₂ = E or D _X3 = Tou R X ₄ = Q or P X ₅ = S, A or V X ₆ = Q or K , _X7 = V or 1X ₈ = n, A, Y, W, S, T, 1 or V X ₉ = A, N, 1, L, T or V Xio = Q or D Xn = S or T X12 = K or T Xi3 = N or S Xu = F or V Xi5 = G or A Xi6 = A or Q Xi7 = T or R
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description SEQ ID NO: SEQUENCE Xis = L or 1 31C6 L - HUMANIZED VL CONSENSUS 172 DIQMTQSPSSLSASVGDRVTITCRASEHIY SYLSWYQQKPGKXiPKLLIY X2X ₃ KTLAEGVPSRFSGSGSGTX ₄ FTLTISS LQP and DXsATYYCQHHFGSPLTFG QGTR LE IK Xi = A or V _X2 = N, A, Y, W, S, T, 1, or V _X3 = A, C, 1, G, T or V X ₄ = D or QX ₅ = F or V 31C6 H -CDR3 CONSENSUS 173 GGPYGXYFDVXis = W, A, D, E, F, G, 1, K, N, Q, R, S, T, Vor Y 31C6 H -CDR3 VARIANT 174 GGPYGAYFDV 31C6 H -CDR3 VARIANT 175 GGPYGDYFDV 31C6 H -CDR3 VARIANT 176 GGPYGEYFDV 31C6 H -CDR3 VARIANT 177 GGPYGFYFDV 31C6 H -CDR3 VARIANT 178 GGPYGGYFDV 31C6 H -CDR3 VARIANT 179 GGPYGIYFDV 31C6 H -CDR3 VARIANT 180 GGPYGKYFDV 31C6 H -VARIANT CDR3 181 GGPYGNYFDV 31C6 H -CDR3 182 GGPYGQYFDV VARIANT 183 GGPYGRYFDV
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description SEQ ID NO: SEQUENCE 31C6 H -CDR3 184 GGPYGSYFDV VARIANT 185 GGPYGTYFDV 31C6 H -CDR3 186 GGPYGVYFDV VARIANT 187 GGPYGYYFDV
[0124] In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof comprises three CDRs of the light chain CDRL1, CDRL2 and CDRL3 and / or three CDRs of the heavy chain CDRH1, CDRH2 and CDRH3.
[0125] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising SEQ ID NO: 35, a CDRH2 comprising SEQ ID NO: 36, a CDRH3 comprising any of SEQ ID NOs: 37, 103, 104, 105, 106, 107 or 160, a CDRL1 comprising SEQ ID NO: 38, a CDRL2 comprising any of SEQ ID NOs: 39, 89, 90, 91, 92, 93, 94 , 95, 96, 97 or 69, and a CDRL3 comprising any of SEQ ID NOs: 40, 98, 99, 100, 101, 102 or 162.
[0126] In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising SEQ ID NO: 81, a CDRH2 comprising SEQ ID NO: 82, a CDRH3 comprising SEQ ID NO: 83, a CDRL1 comprising SEQ ID NO: 84, a CDRL2 comprising SEQ ID NO: 85 and a CDRL3 comprising SEQ ID NO: 86.
[0127] In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising SEQ ID NO: 108, a CDRH2 comprising any of SEQ ID NOs: 109, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 154, 155 or 167, a CDRH3 comprising one of SEQ ID NOs: 110, 173,
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174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 or 187, a CDRL1 comprising SEQ ID NO: 111, a CDRL2 comprising any of SEQ ID NOs: 112 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 or 168 and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 113.
[0128] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising SEQ ID NO: 35, a CDRH2 comprising SEQ ID NO: 36, a CDRH3 comprising SEQ ID NO: 37 , a CDRL1 comprising SEQ ID NO: 38, a CDRL2 comprising SEQ ID NO: 39 and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 40.
[0129] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises CDRH1 comprising SEQ ID NO: 108, a CDRH2 comprising any of SEQ ID NO: 109, 154 or 145, a CDRH3 which comprises SEQ ID NO: 110, a CDRL1 comprising SEQ ID NO: 111, a CDRL2 comprising SEQ ID NO: 112 and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 113.
[0130] In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 108, a CDRH2 comprising SEQ ID NO: 154, a CDRH3 which comprises SEQ ID NO: 110, a CDRL1 comprising SEQ ID NO: 111, a CDRL2 comprising SEQ ID NO: 112 and a CDRL3 comprising SEQ ID NO: 113.
[0131] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain and a variable region of the light chain. In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region
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[0132] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising SEQ ID NO: 87 and a variable region of the light chain comprising SEQ ID NO: 88.
[0133] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 114 and a light chain variable region comprising SEQ ID NO: 115.
[0134] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising any of SEQ ID NOs: 43-58, 65-75 and 87 and a variable region of light chain comprising any of SEQ ID NOs: 59-64, 76-80 and 88.
[0135] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising any of SEQ ID NOs: 144-149 and a variable region of the light chain comprising any of SEQ ID NOs: 150-153.
[0136] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising a variable region of the heavy chain comprising SEQ ID NO: 148 and a variable region of the light chain which comprises SEQ ID NO: 152.
[0137] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 147 and a light chain variable region comprising SEQ ID NO: 150.
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[0138] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising SEQ ID NO: 148 and a variable region of the light chain comprising SEQ ID NO: 153.
[0139] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising SEQ ID NO: 163 and a variable region of the light chain comprising SEQ ID NO: 165.
[0140] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 169 and a light chain variable region comprising SEQ ID NO: 171.
[0141] In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 164 and a light chain variable region comprising SEQ ID NO: 166.
[0142] In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 170 and a light chain variable region comprising SEQ ID NO: 172.
TABLE 5: Exemplifying sequences of anti-TIGIT antibodies
description SEQ ID NO SEQUENCE 14D7 H -CDR1 188 GAWMD 14D7 H -CDR2 189 EIRTKVNNHATNYGESVKG 14D7 H -CDR3 190 ALYDGFYFDY 14D7 L - 191 SASSSVSSGYLY
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description SEQ. ID NO SEQUENCE CDR1 14D7 L -CDR2 192 GTSTLAS 14D7 L -CDR3 193 HQWSSFPYT 14D7 VHPARENTAL 194 EVKLE ESGGG LVQPGGSM KLSCVASG FTFSG AWM DWVRQSP AND KG LEWVAEIRTKVNNHATNYGESVKGR FTIS R D DS KSSVY LQ Μ N N LRAE DSGIYYCRGALYDGFYFDYWG QGTTLTVSS 14D7 VLPARENTAL 195 IQ VLTQSPAIMSASPG EKVN LTCSASSSVSSGYLYWYQQKPGSS PKLWIYGTSTLASGVPARFSGSGSGTSYSLTISNMEAEDAASYF CHQWSSFPYTFGG GTKLE Μ K Hul4D7 VH consensus sequence humanizes the 196 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAX1X2DWVRQAP g kg LEWVAEIRTKVN C Η ATN YG ESVKG RFTISRDX3SKX4X5V YLQX6X7X8LRAEDX9AVYYCRGALYX10X11FYFDYWGQGTLVT VSS Xi = W, A, R, N, D, Q, E, G, Η, 1, L, K, M, P, S, T, Y, V _X2 = Μ, V, L, 1, G, A, S, TX ₃ = D, A, R, N, Q, E, G, Η, 1, L, K, F, S, T, Y, V X ₄ = S, NX ₅ = T, S X ₆ = M, L X ₇ = N, S X ₈ = S, N X ₉ = T, s Xio = D, A, R, N, Q, E, G, Η, 1, L , K, F, P, S, T, W, Y, V Xii = G, A, R, N, D, Q, E, Η, 1, L, K, F, P, S, T, W, Y, V Hul4D7 VH1 (humanized VH chain) 197 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEI RTKVN NHATNYGESVKGRFTISRDDSKSTVYL QMNSLRAEDTAVYYCRGALYDGFYFDYWGQGTLVTVSS Hul4D7 VH2 (humanized VH chain) 198 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAP G KG LEWVAEI RTKVN NHATNYGESVKGRFTIS R D DS KSSVY L QMNSLRAEDTAVYYCRGALYDGFYFDYWGQGTLVTVSS Hul4D7 199 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAP
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description SEQ. ID NO SEQUENCE VH3 (humanized VH chain)G KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKNTVYLQM NSLRAEDTAVYYCRGALYDGFYFDYWG QGTLVTVSS Hul4D7 VL Consensus sequence humanizes the 200 EIVLTQSPATLSLSPGERAXiLSCSASSSVSSGYLYWYQQKPGQ APX7LX2IYGTSTLASGX8PARFSGSGSGTDYTLTISSX3EPEDX4A VYYCHQXsSSFPYTFGQGTKLEXgK Xi = T, S, X ₂ = W, A, R, N, D, Q, E, G, Η, 1, L, K, M, P, S, T, Y, V, _X3 = L, V , 1 X ₄ = F, V, L, 1, T X ₅ = W, A, R, N, D, Q, E, G, Η, 1, L, K, F, P, S, T, Y, VX ₆ = 1, L X ₇ = K, R X ₈ = V, 1 HU14D7V LI (humanized VL chain) 201 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQWSSFPYTFGQGTKLEIK HU14D7V L2 (humanized VL chain) 202 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSFPYTFGQGTKLEI K HU14D7V L3 (humanized VL chain) 203 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSFPYTFGQGTKLEIK 26B10 H -CDR1 204 EFTMH 26B10 H -CDR2 205 GLKPDNGGISYNQKFKG 26B10 H -CDR3 206 GAYYRYDADY 26B10 L - 207 KASQDVKTAVA
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description SEQ. ID NO SEQUENCE CDR1 26B10 L-CDR2 208 SASYRNT 26B10 L-CDR3 209 QQHYSTPFT 26B10 PARENTAL VH 210 EVQLQQSGPELVKPGASVKISCKTSGYTFTEFTMHWVKQSHGKSLEWIGGLKPDNGGISYNQKFKGRATLAVDKSSNTAYMELRSLTSEDSAVYYCARGAYYRYDADYWGQGTTLTVSS 26B10 PARENTAL VL 211 D1VLTQS H KF M STSVG D RVSITCKASQDVKTAVAWYQQKSG Q SPKLLIYSASYRNTGVPDRFTGSGSGTDFTFTIDSVQAEDLAVY FCQQHYSTPFTFGTGTKLELK 26B10 VH Sequence CONSENS 0 HUMANIZ ADA 212 EVQLVQSGAEVKKPGASVKISCKX1SGYTFTEFTX2HWVX3QAP g kg LEWIGGLKPDX4X5GISYNQKFKGRATLTVDX6STX7TAYX8 ELSSLRSEDX9AVYYCARGAYYRYX10X11DYWGQGTLVTVSS Xi = T, V _X2 = Μ, V, G 1, G, A, S, T, _X3 = K, R _X4 = N, A, R, D, Q, E, G, Η, 1, L, K, F, P, S, T, W, Y, VX ₅ = G, A, R, N, D, Q, E, Η, 1, L, K, F, P, S, T , W, Y, V Xê = k, t, d, s X ₇ = N, S X ₈ = Μ, V, L, 1, G, A, S, T X ₉ = T, S Xio = D, A, R , N, Q, E, G, Η, 1, L, K, F, P, S, T, W, Y, V χ _{11 =} a, R, N, D, Q, E, G, Η, 1 , L, K, F, P, S, T, W, Y, V, M Hu26B10 VH1 (humanized VH chain) 213 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPGKG LEWIGGLKPDNGGISYNQKFKGRATLTVDKSTNTAYMELS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH2 (humanized VH chain) 214 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDNGGISYNQKFKGRATLTVDKSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 215 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPG
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description SEQ. ID NO SEQUENCE VH3 (humanized VH chain)KG LEWIGGLKPDNGGISYNQKFKGRATLTVDTSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS 26B10 VL Sequence CONSENS 0 HUMANIZ ADA 216 DIQLTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKA PKLLIYSASYRX1X2GVP X ₃ R FSG SG SGTD FTX4TI SS LQP EDF ATYYCQQHYSTPFTFG QGT KLEIK Xi = N, Q, D, T and X ₂ = S, X ₃ = D, S, _X4 = F, L Hu26B10 VL1 (humanized VL chain) 217 DIQLTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPG KA PKLLIYSASYRNTGVPDRFSGSGSGTDFTFTISSLQPEDFATYYC QQHYSTPFTFGQGTKLEIK Hu26B10 VL2 (humanized VL chain) 218 D1QLTQS PSS LSASVG D RVTITCKASQDVKTAVAWYQQKPG K APKLLIYSASYRNTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYC QQHYSTPFTFGQGTKLEIK Hu26B10 VL3 (humanized VL chain) 219 DIQLTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPG KAPKLLIYSASYRNTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPFTFGQGTKLEIK 14D7 H -CDR3 (D104E) 220 ALYEGFYFDY 14D7 H -CDR3 (G105A) 221 ALYDAFYFDY 14D7 H -CDR3 (G105S) 222 ALYDSFYFDY Hul4D7 223 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAP
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description SEQ. ID NO SEQUENCE VH1 (humanized VH chain) (D104E)GKGLEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSTVYLQMNSLRAEDTAVYYCRGALYEGFYFDYWGQGTLVTVSS Hul4D7 VH1 (humanized VH chaina) (G105A) 224 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSTVYL QMNSLRAEDTAVYYCRGALYDAFYFDYWGQGTLVTVSS Hul4D7 VH1 (humanized VH chaina) (G105S) 225 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSTVYL QMNSLRAEDTAVYYCRGALYDSFYFDYWGQGTLVTVSS Hul4D7 VH2 (humanized VH chain) (D104E) 226 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSSVYL QMNSLRAEDTAVYYCRGALYDEFYFDYVVGQGTLVTVSS Hul4D7 VH2 (humanized VH chain) (G105A) 227 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSSVYL QMNSLRAEDTAVYYCRGALYDAFYFDYWGQGTLVTVSS Hul4D7 VH2 (humanized VH chain) (G105S) 228 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKSSVYL QMNSLRAEDTAVYYCRGALYDSFYFDYWGQGTLVTVSS Hul4D7 VH3 (humanized VH chain) (D104E) 229 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKNTVYL QM NSLRAEDTAVYYCRGALYEGFYFDYWG QGTLVTVSS Hul4D7VH3 230 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKNTVYL
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description SEQ. ID NO SEQUENCE (humanized VH chain) (G105A)QM NSLRAEDTAVYYCRGALYDAFYFDYWG QGTLVTVSS Hul4D7 VH3 (humanized VH chain) (G105S) 231 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGAWMDWVRQAPG KG LEWVAEIRTKVNNHATNYGESVKGRFTISRDDSKNTVYL QM NSLRAEDTAVYYCRGALYDSFYFDYWG QGTLVTVSS 14D7 L -CDR3 (W92A) 232 HQASSFPYT 14D7 L CDR3 (W9 2D) 233 HQDSSFPYT 14D7 L CDR3 (W9 2E) 234 HQESSFPYT 14D7 L CDR3 (W9 2F) 235 HQFSSFPYT 14D7 L CDR3 (W9 2G) 236 HQGSSFPYT 14D7 L -CDR3 (W92H) 237 HQHSSFPYT HU14D7V LI (humanized VL chain) (W92A) 238 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQASSFPYTFGQGTKLEIK HU14D7VLI (string 239 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY
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description SEQ. ID NO SEQUENCE Humanized VL) (W92D)CHQDSSFPYTFGQGTKLEIK HU14D7V LI (humanized VL chain) (W92E) 240 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQESSFPYTFGQGTKLEIK HU14D7V LI (humanized VL chain) (W92F) 241 E1VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPGQA PKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQFSSFPYTFGQGTKLEIK HU14D7V LI (humanized VL chain) (W92G) 242 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQGSSFPYTFGQGTKLEIK HU14D7V LI (humanized VL chain) (W92H) 243 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PKLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQHSSFPYTFGQGTKLEIK HU14D7V L2 (humanized VL chain) (W92A) 244 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQASSFPYTFGQGTKLEIK HU14D7VL2 (stringVL humanizes 245 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQDSSFPYTFGQGTKLEIK
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description SEQ. ID NO SEQUENCE da) (W92D) HU14D7V L2 (humanized VL chain) (W92E) 246 E1VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQESSFPYTFGQGTKLEIK Hul4D7V L2 (humanized VL chain) (W92F) 247 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQFSSFPYTFGQGTKLEIK Hul4D7V L2 (humanized VL chain) (W92G) 248 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQGSSFPYTFGQGTKLEIK Hul4D7V L2 (humanized VL chain) (W92H) 249 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYY CHQHSSFPYTFGQGTKLEIK Hul4D7V L3 (humanized VL chain) (W92A) 250 E1 VLTQSPATLSLSPG AND RATLSCSASSSVSSGYLYWYQQKPG QA PRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQASSFPYTFGQGTKLEIK Hul4D7V L3 (humanized VL chain) (W92D) 251 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQDSSFPYTFGQGTKLEIK
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description SEQ. ID NO SEQUENCE HU14D7V L3 (humanized VL chain) (W92E) 252 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQESSFPYTFGQGTKLEIK HU14D7V L3 (humanized VL chain) (W92H) 253 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQFSSFPYTFGQGTKLEIK HU14D7V L3 (humanized VL chain) (W92G) 254 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQGSSFPYTFGQGTKLEIK HU14D7V L3 (humanized VL chain) (W92H) 255 EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGQAPRLWIYGTSTLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQHSSFPYTFGQGTKLEIK 26B10 H -CDR2 (N55Q) 256 GLKPDQGGISYNQKFKG 26B10 H CDR2 (N55 D) 257 GLKPDDGGISYNQKFKG 26B10 H CDR2 (N56 A) 258 GLKPDNAGISYNQKFKG 26B10 H CDR2 (N55 T) 259 GLKPDTGGISYNQKFKG 26B10 H CDR2 (N55 260 GLKPDSGGISYNQKFKG
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description SEQ. ID NO SEQUENCE S) 26B10 H CDR2 (N55 G) 261 GLKPDGGGISYNQKFKG 26B10 H -CDR2 (G56S) 262 GLKPDNSGISYNQKFKG 26B10 H CDR2 (G56 T) 263 GLKPDNTGISYNQKFKG Hu26B10 VH1 (VH chain humanizesda) (N55Q 264 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPG KG LEWIGGLKPDOGGISYNQKFKGRATLTVDKSTNTAYME LS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (N55D) 265 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPG KG LEWIGGLKPDDGGISYNQKFKGRATLTVDKSTNTAYM AND LS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (G56A) 266 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPGKG LEWIGGLKPDNAGISYNQKFKGRATLTVDKSTNTAYMELS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH1 (VH chain humanizesda) (N55T) 267 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPG KG LEWIGGLKPDTGGISYNQKFKGRATLTVDKSTNTAYM AND LS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (N55S) 268 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPGKG LEWIGGLKPDSGGISYNQKFKGRATLTVDKSTNTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 269 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPGKG LEWIGGLKPDGGGISYNQKFKGRATLTVDKSTNTAYMELS
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description SEQ. ID NO SEQUENCE (humanized VH chain) (N55G)SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (G56S) 270 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPGKG LEWIGGLKPDNSGISYNQKFKGRATLTVDKSTNTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (G56T) 271 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVKQAPG KG LEWIGGLKPDNTGISYNQKFKGRATLTVDKSTNTAYM AND LS SLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH2 (humanized VH chain) (N55Q) 272 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDQGGISYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH2 (humanized VH chain) (N55D) 273 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDDGGISYNQKFKGRATLTVDKSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH1 (humanized VH chain) (G56A) 274 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDNAGISYNQKFKGRATLTVDKSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH2 (humanized VH chain) (N55T) 275 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDTGGISYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVS S Hu26B10VH2 276 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDSGGISYNQKFKGRATLTVDKSTSTAYM AND LSS
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description SEQ. ID NO SEQUENCE (humanized VH chain) (N55S)LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10VH2 (humanized VH chain) (N55G) 277 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDGGGISYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH2 (humanized VH chain) (G56S) 278 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDNSGISYNQKFKGRATLTVDKSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH2 (humanized VH chain) (G56T) 279 EVQLVQSGAEVKKPGASVKISCKTSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDNTGISYNQKFKGRATLTVDKSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (N55Q) 280 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDQGGISYNQKFKGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (N55D) 281 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDDGGISYNQKFKGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (G56A) 282 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDNAGISYNQKFKGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 283 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPG
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description SEQ ID NO SEQUENCE VH3 (humanized VH chain) (N55T)KG LEWIGG LKPDTGGISYNQKFKGRATLTVDTSTSTAYM ELSS LRS AND DTAVYYCARGAYYRYDADYWG QGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (N55S) 284 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPGKG LEWIGGLKPDSGGISYNQKFKGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (N55G) 285 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPG KG LEWIGG LKPDGGG ISYNQKFKGRATLTVDTSTSTAYM AND LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (G56S) 286 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPGKG LEWIGG LKPDNSG ISYNQKFKGRATLTVDTSTSTAYM ELSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS Hu26B10 VH3 (VH chain humanizesda) (G56T) 287 EVQLVQSGAEVKKPGASVKISCKVSGYTFTEFTMHWVRQAPG KG LEWIGGLKPDNTGISYNQKFKGRATLTVDTSTSTAYME LSS LRSEDTAVYYCARGAYYRYDADYWGQGTLVTVSS hTIGIT epitope (24-41) 288 SSTTAQVNWEQQDQL hTIGIT epitope (85-93) 289 IYHTYPDGT hTIGIT epitope (96-100) 290 GRIFL
[0143] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment comprises a CDRH1 comprising SEQ ID NO: 188, a
CDRH2 comprising SEQ ID NO: 189, a CDRH3 comprising any
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88/145 one of SEQ ID NOs: 190, 220, 221 or 222, a CDRL1 comprising SEQ ID NO: 191, a CDRL2 comprising SEQ ID NO: 192 and a CDRL3 comprising any of SEQ ID NOs: 193, 232, 233, 234, 235, 236 or 237.
[0144] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a CDRH1 comprising SEQ ID NO: 204, a CDRH2 comprising any of SEQ ID NOs: 205, 256, 257, 258, 259, 260, 261, 262 or 263, a CDRH3 comprising SEQ ID NO: 206, a CDRL1 comprising SEQ ID NO: 207, a CDRL2 comprising SEQ ID NO: 208 and a CDRL3 comprising SEQ ID NO: 209.
[0145] In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable region of the heavy chain and a variable region of the light chain. In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising SEQ ID NO: 194 and a variable region of the light chain comprising SEQ ID NO: 195.
[0146] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 196 and a light chain variable region comprising SEQ ID NO: 200.
[0147] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 210 and a light chain variable region comprising SEQ ID NO: 211.
[0148] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising SEQ ID NO: 212 and a variable region of the light chain comprising SEQ ID NO: 216.
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[0149] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising any of SEQ ID NOs: 197, 198, 199, 223, 224, 225, 226, 227, 228, 229, 230 and 231 and a variable light chain region comprising any of SEQ ID NOs: 201, 202, 203, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 , 248, 249, 250, 251, 252, 253, 254 and 255.
[0150] In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain comprising any of SEQ ID NOs: 213, 214, 215, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285 and 286 and a variable light chain region comprising any of the SEQs ID NOs: 217, 218 and 219.
[0151] Additional anti-TIGIT antibodies that can be used in the formulations in the present invention described include those disclosed, for example, in International PCT Application No. WO 2016/106302; WO 2016/011264; and WO 2009/126688.
TABLE 6: Exemplary heavy chain sequences
IgGl heavy chain constant domain 291 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG N VFSCSVM H EALH N H YTQKSLSLSPG K Heavy chain constant domain lgG4 S228P 292 TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSN KG LPSSIE KTISKA
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Kappa light chain constant domain 293 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0152] In any of the embodiments mentioned above, the anti-TIGIT antibody or antigen-binding fragment thereof is an antibody comprising any of the variable heavy chains described above and any constant domain of the human heavy chain. In one embodiment, the antibody or antigen-binding fragment thereof is of the IgG isotype and comprises a human constant domain of the human IgG1, IgG2, IgG3 or IgG4 heavy chain. In one embodiment, the antibody or antigen binding fragment thereof comprises a human IgG1 heavy chain constant domain (SEQ ID NO: 291) or a variant thereof, wherein the variant comprises up to 20 modified amino acid substitutions . In one embodiment, the antibody or antigen-binding fragment thereof is an antibody that comprises a human IgGl heavy chain constant domain that comprises the amino acid sequence of SEQ ID NO: 291. In one embodiment, the antibody or The antigen-binding fragment of the invention comprises a human IgGl heavy chain constant domain in which the IgGl constant domain is afucosylated. In one embodiment, the antibody or antigen-binding fragment thereof comprises a constant domain of the human IgG4 heavy chain or a variant thereof, wherein the variant comprises up to 20 modified amino acid substitutions. In another embodiment, the antibody or antigen-binding fragment of the invention comprises a constant domain of the human IgG4 heavy chain, where the amino acid at position 228 (using the EU numbering scheme) has been replaced from Ser to Pro In one mode, the
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The antibody or antigen-binding fragment of the invention comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292.
[0153] In any of the modalities mentioned above, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise any of the variable light chains described above and the constant domain of the human light chain. In one embodiment, the antibody or antigen-binding fragment thereof comprises a human kappa light chain constant domain or a variant thereof, wherein the variant comprises up to 20 modified amino acid substitutions. In another embodiment, the antibody or antigen-binding fragment thereof comprises a human lambda light chain constant domain or a variant thereof, wherein the variant comprises up to 20 modified amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment of the invention comprises a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293.
Formulations
[0154] The formulations of the invention minimize the formation of antibody aggregates (high molecular weight species) and particles, high and low molecular weight species, minimize the oxidation of methionine residues and ensure that the antibody retains biological activity throughout time.
[0155] In one aspect, the present invention includes various formulations of an anti-TIGIT antibody or antigen-binding fragment thereof. For example, the present invention includes formulations comprising (i) an anti-TIGIT antibody or antigen-binding fragment thereof, (ii) a buffer (for example, L-histidine or acetate), (iii) a non-reducing sugar (per
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92/145 (example, sucrose); (iv) a non-ionic surfactant (for example, polysorbate 80); and (v) an antioxidant (for example, L-methionine). In one embodiment, the formulation further comprises an anti-PD1 antibody. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μΜ to about 50 μΜ. In one embodiment, the chelator is diethylenetriaminopentacetic acid (DTPA). In another embodiment, the chelator is EDTA.
[0156] In another aspect, the present invention also includes various co-formulations of an anti-TIGIT antibody or antigen binding fragment thereof and a human anti-PD-1 antibody or antigen binding fragment thereof. In one embodiment, the formulation comprises (i) an anti-TIGIT antibody or antigen-binding fragment thereof, (ii) an anti-human PD-1 antibody or antigen-binding fragment thereof, (iii) a buffer ( for example, L-histidine or acetate), (iv) a non-reducing sugar (for example, sucrose), (v) a non-ionic surfactant (for example, polysorbate 80) and (vi) an antioxidant (for example, L- methionine). In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μΜ to about 50 μΜ. In one embodiment, the chelator is diethylenetriaminopentacetic acid (DTPA). In another embodiment, the chelator is EDTA.
[0157] The pharmaceutical formulations of the present invention can include buffers.
[0158] Buffers that are useful in the pharmaceutical formulations and methods of the invention include succinate (sodium or potassium), L-histidine, phosphate (sodium or potassium), Tris (tris (hydroxymethyl) aminomethane), diethanolamine, citrate (sodium), acetate (sodium) and the like. In one embodiment of the invention, the buffer is present in the formulation at a concentration of about 1 to 20 mM (1, 2, 3, 4,
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5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mM). In specific embodiments of the invention, the buffer is histidine, for example, Lhistidine buffer.
[0159] The buffer of this invention has a pH in the range of about 4.5 to about 6.5; about 5.0 to 6.2; about 5.5 to 6.0; and preferably has a pH of about 5.8. To arrive at the example formulation, histidine and acetate buffers in the pH range of 5.0 to 6.0 were explored for adequacy. When a range of pH values is recited, such as a pH between pH 5.5 and 6.0, the range should include the recited values. For example, a range of about 5.0 to about 6.0 includes 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0. Unless otherwise indicated, pH refers to pH after reconstitution of the lyophilized formulations of the present invention. The pH is typically measured at 25 ° C using the standard glass bulb pH meter. As used in the present invention, a solution comprising histidine buffer at pH X refers to a solution at pH X and which comprises histidine buffer, that is, the pH is intended to refer to the pH of the solution.
[0160] In an embodiment of the invention, the anti-TIGIT formulation and the co-formulation of anti-human TIGIT and anti-PD-1 comprise a non-reducing sugar. As used herein, non-reducing sugar is sugar that is not capable of acting as a reducing agent because it does not contain or cannot be converted to contain a free aldehyde group or a free ketone group. Examples of non-reducing sugars include, but are not limited to, disaccharides, such as sucrose and trehalose. In one embodiment of the invention, non-reducing sugar is present in an amount of about 1 to 10% (w / v) (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). In another embodiment, non-reducing sugar is present in an amount of about 6% to about 8% (w / v) (6, 7 or 8%). In another modality, non-reducing sugar is present in a
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94/145 amount of about 6% (w / v). In another embodiment, non-reducing sugar is present in an amount of about 7% (w / v). In another embodiment, non-reducing sugar is present in an amount of about 8% (w / v). In one embodiment, sucrose, trehalose or raffinose do not reduce sugar. In another embodiment, non-reducing sugar is sucrose. In another embodiment, sucrose is present in 6 to 8% w / v. In one embodiment, sucrose is present in about 6% (w / v). In one embodiment, sucrose is present in about 7% (w / v). In one embodiment, sucrose is present in about 8% (w / v).
[0161] The formulations of the invention also comprise a surfactant. As in the present invention used, a surfactant is a surface active agent that is amphipathic in nature. Surfactants can be added to the formulations in this document to provide stability, reduce and / or prevent aggregation or to prevent and / or inhibit damage to proteins during processing conditions, such as purification, filtration, lyophilization, transport, storage and delivery. In the present invention, a surfactant can be useful to provide additional stability to the active ingredient (or active ingredients).
[0162] Nonionic surfactants that may be useful in formulations, including the co-formulation of the invention, include, among others, esters of polyoxyethylene sorbitan fatty acids (Polysorbates, sold under the trade name Tween® (Uniquema Americas LLC, Wilmington, DE )) including Polysorbate 20 (polyoxyethylene sorbitan monolaurate), Polysorbate 40 (polyoxyethylene sorbitan monopalmitate), Polysorbate 60 (polyoxyethylene sorbitan monostearate) and Polysorbate 80 (polyoxyethylene sorbitan monooleate); alkyl polyoxyethylene ethers such as Brij® 58 (Uniquema Americas LLC, Wilmington, DE) and Brij® 35; poloxamers (for example, poloxamer 188); Triton® X-100 (Union
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Carbide Corp., Houston, TX) and Triton® X-114; NP40; Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; ethylene and propylene glycol copolymers (for example, the pluronic® series of nonionic surfactants, such as pluronic® F68, pluronic® 10R5, pluronic® F108, pluronic® F127, pluronic® F38, pluronic® L44, pluronic® L62 (BASF Corp. , Ludwigshafen, Germany) and sodium dodecyl sulfate (SDS) In one embodiment, the nonionic surfactant is polysorbate 80 or ο polysorbate 20. In one embodiment, the nonionic surfactant is polysorbate 20. In another embodiment, the surfactant non-ionic is polysorbate 80.
[0163] The amount of non-ionic surfactant to be included in the formulations of the invention is an amount sufficient to perform the desired function, that is, the minimum amount necessary to stabilize the active pharmaceutical ingredient (i.e., the anti-TIGIT antibody or fragment binding to the antigen thereof, or the anti-TIGIT antibody or antigen binding fragment thereof and the anti-human PD-1 antibody or antigen binding fragment thereof) in the formulation. All percentages for the nonionic surfactant are listed in% w / v. Typically, the surfactant is present in a concentration of about 0.008% to about 0.1% w / v. In some embodiments of this aspect of the invention, the surfactant is present in the formulation in an amount of about 0.01% to about 0.1%; from about 0.01% to about 0.09%; from about 0.01% to about 0.08%; from about 0.01% to about 0.07%; from about 0.01% to about 0.06%; from about 0.01% to about 0.05%; from about 0.01% to about 0.04%; from about 0.01% to about 0.03%, from about 0.01% to about 0.02%, from about 0.015% to about 0.04%; from about 0.015% to about 0.03%, from about 0.015% to about 0.02%, from about 0.02% to about 0.04%, from about 0.02% to about from 0.035% or from about 0.02% to about 0.03%. In specific modalities, the surfactant is present in an amount of about 0.02%. In alternative modalities,
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96/145 the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035% or about 0.04%.
[0164] In exemplary embodiments of the invention, the surfactant is a nonionic surfactant selected from the group consisting of: Polysorbate 20 and Polysorbate 80. In preferred embodiments, the surfactant is Polysorbate 80.
[0165] In specific embodiments, the formulations, including co-formulations, of the invention comprise about 0.01% to about 0.04% w / v of polysorbate 80. In other embodiments, the formulations in the present invention described comprise polysorbate 80 in an amount of about 0.008% w / v, about 0.01% w / v. In one embodiment, the amount of polysorbate 80 is about 0.015% w / v. In another embodiment, the amount of polysorbate 80 is about 0.02% w / v. In another embodiment, the amount of polysorbate 80 is about 0.025% w / v. In another embodiment, the amount of polysorbate 80 is about 0.03% w / v. In another embodiment, the amount of polysorbate 80 is about 0.035% w / v. In another embodiment, the amount of polysorbate 80 is about 0.04% w / v. In another embodiment, the amount of polysorbate 80 is about 0.045% w / v. In particular embodiments, the formulations of the invention comprise about 0.02% w / v of polysorbate 80.
[0166] The formulations, including co-formulations, of the present invention also comprise methionine, or its pharmaceutically acceptable salt, as an antioxidant. In one embodiment, methionine is L-methionine. In another embodiment, methionine is a pharmaceutically acceptable salt of Lmethionine, such as, for example, methionine HCL. In one embodiment of the invention, methionine is present in the formulation at a concentration of about 1 to 20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 and 20 mM). In another embodiment, methionine is present at about 5 mM
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97/145 at about 10 mM (5, 6, 7, 8, 9 and 10 mM). In another embodiment, methionine is present in about 10 mM.
[0167] The formulations, including co-formulations, of the present invention can also further comprise a chelating agent. In one embodiment of the invention, the chelating agent is present in the formulation at a concentration of about 5 to 30 μΜ (for example, 5, 10, 15, 20, 25 or 30 μΜ). In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA.
Freeze-dried pharmaceutical compositions
[0168] Lyophilized therapeutic protein formulations provide several advantages.
[0169] Lyophilized formulations in general offer better chemical stability than solution formulations and, thus, increased half-life. A lyophilized formulation can also be reconstituted in different concentrations, depending on clinical factors, such as route of administration or dosage. For example, a lyophilized formulation can be reconstituted in a high concentration (ie, in a small volume) if necessary for subcutaneous administration or in a lower concentration if administered intravenously. High concentrations may also be necessary if high dosage is required for a particular individual, particularly if administered subcutaneously where the injection volume should be minimized. One such lyophilized antibody formulation is disclosed in US patent 6,667,958, which is incorporated by reference in its entirety by reference. Lyophilized formulations of another therapeutic protein are disclosed in US patent 7,247,707, which is incorporated herein by reference in its entirety.
[0170] Typically, the lyophilized formulation is prepared in anticipation of
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98/145 reconstitution in high concentration of medication (DP, in an exemplary form of humanized anti-PD-1 antibody of pembrolizumab, or antigen-binding fragment thereof), that is, in anticipation of reconstitution in a low volume of water. Subsequent dilution with water or isotonic buffer can be readily used to dilute the DP to a lower concentration. Typically, excipients are included in a lyophilized formulation of the present invention at levels that will result in an approximately isotonic formulation when reconstituted in a high concentration of DP, for example, for subcutaneous administration. Reconstitution in a larger volume of water to obtain a lower concentration of DP will necessarily reduce the tonicity of the reconstituted solution, but this reduction may have little significance in non-subcutaneous, for example, intravenous administration. If isotonicity is desired in a lower concentration of DP, the lyophilized powder can be reconstituted in the low standard volume of water and then diluted with isotonic diluent, such as 0.9% sodium chloride.
[0171] The lyophilized formulations of the present invention are formed by lyophilization (lyophilization) of a pre-lyophilization solution. Lyophilization is carried out by freezing the formulation and subliming the water to a temperature suitable for primary drying. Under this condition, the temperature of the product is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for primary drying ranges from about -30 to 25 ° C (as long as the product remains frozen during primary drying) at an appropriate pressure, typically ranging from about 50 to 250 mTorr. The formulation, size and type of the container containing the sample (for example, glass bottle) and the volume of liquid determine the time needed for drying, which can vary from a few hours to several days (for example, 40 to 60 hours) ). A secondary drying step can be
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99/145 carried out at about 0 to 40 ° C, depending mainly on the type and size of the container and the type of protein used. The secondary drying time is determined by the desired residual moisture level in the product and typically takes at least about 5 hours. Typically, the moisture content of a lyophilized formulation is less than about 5% and preferably less than about 3%. The pressure can be the same as that used during the primary drying step. Lyophilization conditions may vary, depending on the formulation and size of the bottle.
[0172] In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein reconstitution is to be carried out, in order to avoid a transfer step. The container in this case can, for example, be a 3, 5, 10, 20, 50 or 100 cc bottle.
[0173] The lyophilized formulations of the present invention are reconstituted prior to administration. The protein can be reconstituted at a concentration of about 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL or higher concentrations, such as 150 mg / mL, 200 mg / ml, 250 mg / ml or 300 mg / ml to about 500 mg / ml. In one embodiment, the protein concentration after reconstitution is about 10 to 300 mg / mL. In one embodiment, the protein concentration after reconstitution is about 20 to 250 mg / mL. In one embodiment, the protein concentration after reconstitution is about 150 to 250 mg / mL. In one embodiment, the protein concentration after reconstitution is about 180 to 220 mg / mL. In one embodiment, the protein concentration after reconstitution is about 50 to 150 mg / mL. In one embodiment, the protein concentration after reconstitution is about 100 mg / mL. In one embodiment, the protein concentration after reconstitution is about 75 mg / mL. In one embodiment, the protein concentration after reconstitution is about 50 mg / mL. In one modality, the
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100/145 protein concentration after reconstitution is about 25 mg / mL. High concentrations of protein are particularly useful when subcutaneous release of the reconstituted formulation is desired. However, for other routes of administration, such as intravenous administration, lower concentrations of the protein (for example, from about 5 to 50 mg / ml) may be desired.
[0174] Reconstitution usually takes place at a temperature of around 25 ° C to ensure complete hydration, although other temperatures can be employed as desired. The time required for reconstitution will depend, for example, on the type of diluent, amount of excipient (or excipients) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (for example, phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.
Liquid pharmaceutical compositions
[0175] A liquid antibody formulation can be made by taking the drug substance (for example, humanized anti-PD-1) which is in liquid form (for example, pembrolizumab in an aqueous pharmaceutical formulation) and exchanging it for the buffer desired as the last stage of the purification process. There is no lyophilization step in this modality. The drug substance in the final buffer is concentrated to a desired concentration. Excipients such as sucrose and polysorbate 80 are added to the drug substance and are diluted using the appropriate buffer to the final protein concentration. The final formulated drug substance is filtered using 0.22 pm filters and placed in a final container (for example, glass bottles).
III. Usage Methods
[0176] The invention also relates to a method of treating cancer
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101/145 in an individual, wherein the method comprises administering an effective amount of any of the formulations of the invention; that is, any formulation in the present invention described, for the individual. In some specific embodiments of this method, the formulation is administered to the individual via intravenous administration. In other embodiments, the formulation is administered to the individual by subcutaneous administration. In one embodiment, the invention comprises a method of treating cancer in a human patient which comprises administering any formulation of the invention to the patient.
[0177] In any of the methods of the invention, cancer can be selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma, kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer (for example, refractory prostate adenocarcinoma hormone), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignancies.
[0178] In some modalities, lung cancer in non-small cell lung cancer.
[0179] In alternative modalities, lung cancer is a small cell cancer.
[0180] In some modalities, the lymphoma is Hodgkin's lymphoma.
[0181] In other modalities, lymphoma is a non-Hodgkin's lymphoma. In particular modalities, lymphoma is a large B-cell mediastinal lymphoma.
[0182] In some modalities, breast cancer is breast cancer
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102/145 triple negative.
[0183] In other modalities, breast cancer is ER + / HER2- breast cancer.
[0184] In some modalities, bladder cancer is urothelial cancer.
[0185] In some modalities, head and neck cancer is nasopharyngeal cancer. In some modalities, cancer is thyroid cancer. In other modalities, cancer is salivary cancer. In other modalities, cancer is squamous cell carcinoma of the head and neck.
[0186] In one embodiment, the invention comprises a method of treating non-small cell metastatic lung cancer (NSCLC) in a human patient which comprises administering a formulation of the invention to the patient. In specific modalities, the patient has a tumor with high expression of PD-L1 [(Tumor Ratio Score (TPS)> 50%)]] and has not previously been treated with platinum-containing chemotherapy. In other modalities, the patient has a tumor with PD-L1 expression (TPS> 1%) and has been previously treated with chemotherapy containing platinum. In still other modalities, the patient has a tumor with PD-L1 expression (TPS> 1%) and has not been previously treated with platinum-containing chemotherapy. In specific modalities, the patient had disease progression or after receiving chemotherapy containing platinum. In certain embodiments, the PD-L1 TPS is determined by an FDA approved test. In certain modalities, the patient's tumor does not have EGFR or ALK genomic aberrations. In certain embodiments, the patient's tumor has a genomic aberration of EGFR or ALK and had disease progression after receiving treatment for EGFR or ALK aberrations before receiving the anti-PD-1 antibody or antigen binding fragment.
[0187] In some modalities, cancer is a metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).
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[0188] In some modalities, cancer is a metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).
[0189] In some modalities, cancer is a solid tumor with a high level of microsatellite instability (MSI-H).
[0190] In some modalities, cancer is a solid tumor with a high mutational burden.
[0191] In some modalities, cancer is selected from the group consisting of: melanoma, non-small cell lung cancer, relapsing or refractory classic Hodgkin's lymphoma, head and neck squamous cell carcinoma, urothelial cancer, esophageal cancer, cancer gastric and hepatocellular cancer.
[0192] In other modalities of the above treatment methods, cancer is a heme neoplasm. In certain modalities, the Heme neoplasm is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (LLC), chronic myeloid leukemia (CML), diffuse large B cell lymphoma (DLBCL), DLBCL positive EBV, primary large B cell mediastinal lymphoma, large B cell lymphoma, rich in histiocytes, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), cell leukemia protein 1 (Mcl -1), myelodysplastic syndrome (SMD), non-Hodgkin's lymphoma (NHL) or small lymphocytic lymphoma (SLL).
[0193] Malignancies showing improved disease-free and overall survival in relation to the presence of tumor infiltrating lymphocytes in biopsy or surgical material, for example, melanoma, colorectal, liver, kidney, stomach / esophagus, breast, pancreas and cancer ovary are included in the described methods and treatments in the present invention. These cancer subtypes are known to be susceptible to immune control by T lymphocytes. In addition, refractory or recurrent malignancies are included whose growth
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104/145 can be inhibited using the described antibodies in the present invention.
[0194] Additional cancers that may benefit from treatment with the formulations described in the present invention include those associated with persistent infection by viruses such as the immunodeficiency virus, hepatitis virus class A, B and C, Epstein Barr virus, papilloma virus that are known to cause causation, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervix, vulvar, anal, penis and oral cancer.
[0195] Formulations can also be used to prevent or treat infections and infectious diseases. Thus, the invention provides a method for treating chronic infection in a mammal, which comprises administering an effective amount of a formulation of the invention to the individual. In some specific embodiments of this method, the formulation is administered to the individual via intravenous administration. In other embodiments, the formulation is administered to the individual by subcutaneous administration.
[0196] These agents can be used alone or in combination with vaccines to stimulate the immune response to pathogens, toxins and autoantigens. The antibodies or antigen-binding fragment thereof can be used to stimulate the immune response to infectious viruses in humans, including, but not limited to: human immunodeficiency virus, hepatitis virus class A, B and C, Epstein Barr virus, cytomegalovirus human, human papilloma virus and herpes virus. Antagonistic anti-PD-1 antibodies or antibody fragments can be used to stimulate the immune response to infection by bacterial or fungal parasites and other pathogens. Viral infections with hepatitis B and C and HIV are among those considered to be chronic viral infections.
[0197] The formulations of the invention can be administered to a patient
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105/145 in combination with one or more additional therapeutic agents. The additional therapeutic agent may be a biotherapeutic agent (including, but not limited to, antibodies to VEGF, EGFR, Her2 / neu, VEGF, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4- 1BB and ICOS), an immunogenic agent (for example, attenuated cancer cells, tumor antigens, antigen presenting cells, such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune-stimulating cytokines (for example, IL-2, IFNct2, GM-CSF) and cells transfected with genes encoding immune stimulating cytokines, such as but not limited to GM-CSF).
[0198] As noted above, in some embodiments of the methods of the invention, the method further comprises administering an additional therapeutic agent. In particular embodiments, the additional therapeutic agent is an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-CTL4 antibody or antigen-binding fragment thereof , an anti-CD27 antibody or antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a viral vector for Newcastle disease that expresses IL-12. In another embodiment, the additional therapeutic agent is dinaciclib. In still other embodiments, the additional therapeutic agent is a STING agonist.
[0199] Suitable routes of administration may, for example, include parenteral administration, including intramuscular, subcutaneous, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. Medicines can be administered in several conventional ways, such as intraperitoneal, parenteral, intra-arterial or intravenous injection. The modes of administration in which the volume of the solution should be limited (for example,
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106/145 subcutaneous administration) require a lyophilized formulation to allow reconstitution in high concentration.
[0200] The selection of a dosage of the additional therapeutic agent depends on several factors, including the rate of serum or tissue renewal of the entity, the level of symptoms, the immunogenicity of the entity and the accessibility of target cells, tissues or organs in the subject to to be treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Therefore, the dose amount and dosing frequency of each additional therapeutic agent (for example, biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer being treated and the characteristics of the patient. Guidance on the selection of appropriate doses of antibodies, cytokines and small molecules is available. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, United Kingdom; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348: 601-608; Milgrom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344: 783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 15941602; Physicians 'Desk Reference 2003 (Physicians' Desk Reference, 57 ^ Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). The determination of the appropriate dosage regimen can be made by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment, and will depend, for example, on the patient's clinical history (for example, example, therapy
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107/145), the type and stage of cancer to be treated and biomarkers of response to one or more therapeutic agents in combination therapy.
[0201] Various references from the literature are available to facilitate the selection of pharmaceutically acceptable vehicles or excipients for the additional therapeutic agent. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.
[0202] A pharmaceutical antibody formulation can be administered by continuous infusion or by doses at intervals of, for example, one day, 1 to 7 times a week, one week, two weeks, three weeks, monthly, bimonthly, etc. A preferred dose protocol is one involving the maximum dose or frequency of the dose that avoids significant undesirable side effects. A total weekly dose is generally at least 0.05 pg / kg, 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 100 pg / kg, 0.2 mg / kg kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg of body weight or more. See, for example, Yang et al. (2003) New Engl. J. Med. 349: 427-434; Herold et al. (2002) New Engl. J. Med. 346: 1692-1698; Liu et al. (1999) 7. Neurol. Neurosurg. Psych. 67: 451-456; Portielji etal. (20003) Cancer Immunol. Immunother. 52: 133-144. The desired dose of a small molecule therapy, for example, a
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108/145 peptide mimetic, natural product or organic chemical, is approximately the same as for an antibody or polypeptide, in moles / kg.
[0203] The embodiments of the invention also include one or more of the biological formulations in the present invention described (i) for use in, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicament for: (a) therapy (for example, of the human body); b) medicine; (c) inducing or increasing an anti-tumor immune response (d) decreasing the number of one or more tumor markers in a patient; (e) stop or slow the growth of a tumor or blood cancer; (f) stopping or slowing the progression of a disease related to PD-1 or a disease related to anti-TIGIT; (g) stop or slow progression cancer; (h) stabilization of PD-1-related disease or an anti-TIGIT disease; (i) inhibition of tumor cell growth or survival; (j) eliminate or reduce the size of one or more lesions or cancerous tumors; (k) reducing the progression, onset or severity of PD-1-related disease or an anti-TIGIT disease; (I) reduce the severity or duration of clinical symptoms of PD-1-related or anti-TIGIT-related diseases, such as cancer (m) prolonging a patient's survival in relation to the expected survival in a similar untreated patient n ) induce complete or partial remission of a cancerous condition or other PD-1 or anti-TIGIT related disease, o) cancer treatment, or p) treatment of chronic infections.
GENERAL METHODS
[0204] Standard methods in molecular biology are described by Sambrook, Fritsch and Maniatis (1982 & 1989 2- Edition, 2001 3 ^ Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
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Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^g ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, volume 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, volumes 1-4, John Wiley and Sons, Inc. New York, NY, describing bacterial cell cloning and DNA mutagenesis (Volume 1), cloning in mammalian and yeast cells (volume 2), glycoconjugates and protein expression (Vol.3) and bioinformatics (volume 4).
[0205] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization, are described (Coligan, et al. (2000) Current Protocols in Protein Science, volume 1, John Wiley and Sons, Inc., New York) . Chemical analyzes, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, for example, Coligan, et al. (2000) Current Protocols in Protein Science, volume 2, John Wiley and Sons , Inc., New York; Ausubel, etal. (2001) Current Protocols in Molecular Biology, volume 3, John Wiley and Sons, Inc., NY, NY, pages 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pages 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pages 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, volume 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, for example, Coligan, et al. (2001) Current Protocols in Immunology, volume 4, John Wiley, Inc., New York).
[0206] Monoclonal, polyclonal and humanized antibodies can be
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110/145 prepared (see, for example, Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 139-243; Carpenter et al. (2000) J. Immunol. 165: 6205; He eta /. (1998) 7. Immunol. 160: 1029; Tang eta /. (1999) 7. Biol. Chem. 274: 27371-27378; Baca et al. (1997) J. Biol. Chem. 272: 10678-10684; Chothia et al. (1989) Nature 342: 877-883; Foote and Winter (1992) 7. Mol. Biol. 224: 487-499; Patent 6,329,511).
[0207] An alternative to humanization is to use human antibody libraries displayed on phages or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14: 309314; Barbas (1995) Nature Medicine 1: 837-839 ; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21: 371377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin etal. (1999) Nature Biotechnol. 17: 397-399).
[0208] Antigen purification is not necessary for the generation of antibodies. Animals can be immunized with cells carrying the antigen of interest. Splenocytes can then be isolated from immunized animals, and splenocytes can be fused with a myeloma cell line to produce a hybridoma (see, for example, Meyaard et al. (1997) Immunity 7: 283-290; Wright et al. (2000) Immunity 13: 233-242; Preston et al. Supra; Kaithamana et al. (1999 ^ 7. Immunol. 163: 5157-5164).
[0209] Antibodies can be conjugated, for example, with small molecules of drugs, enzymes, liposomes, polyethylene glycol (PEG). The
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111/145 antibodies are useful for therapeutic, diagnostic, kit or other purposes and include antibodies coupled, for example, to dyes, radioisotopes, enzymes or metals, for example, colloidal gold (see, for example, Le Doussal et al. ( 1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160: 3891-3898; Hsing and Bishop (1999) J. Immunol. 162: 2804-2811; Everts et al. ( 2002) J. Immunol. 168: 883-889).
[0210] Methods for flow cytometry, including fluorescence activated cell classification (FACS), are available (see, for example, Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons , Hoboken, NJ; Givan (2001) Flow Cytometry, 2- Ed .; WileyLiss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides and antibodies, for use, for example, as diagnostic reagents, are available (Molecular Probesy (2003) Catalog, Molecular Probes, Inc., Eugene , OR; Sigma-Aldrich (2003) Catalog, St. Louis, MO).
[0211] Standard methods of histology of the immune system are described (see, for example, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt etal. (2000) Color Atlas of Histology, Lippincott, Williams and Wilkins, Phila, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).
[0212] Software packages and databases for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites and sequence alignments, are available (see, for example, GenBank, Vector NTI ® Suite (Informax, Inc, Bethesda, MD); Wiscosin GCG Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16:
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741-742; Menne et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren et al. (2002) Comput. Biomed Methods Programs. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14: 46834690).
Analytical Methods
[0213] Appropriate analytical methods for evaluating product stability include size exclusion chromatography (SEC), dynamic light scattering test (DLS), differential scanning calorimeter (DSC), iso-asp quantification, power, UV at 340 nm, UV and FTIR spectroscopy. SEC (J. Pharm. Scien., 83: 1645 to 1650, (1994); Pharm. Res. 11: 485 (1994); J. Pharm. Bio. Anal. 15: 1928 (1997); J. Pharm. Bio Anal., 14: 1133 to 1140 (1986)) measures the percentage of monomer in the product and provides information on the amount of soluble aggregates. DSC (Pharm. Res. 15: 200 (1998); Pharm. Res., 9: 109 (1982)) provides information on the protein denaturation temperature and the glass transition temperature. DLS (American Lab., November (1991)) measures the average diffusion coefficient and provides information on the amount of soluble and insoluble aggregates. UV at 340 nm measures the intensity of light scattered at 340 nm and provides information on the amounts of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and provides information on protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45: 231 (1998); Pharm. Res. 12: 1250 (1995); J. Pharm. Scien. 85: 1290 (1996); J. Pharm. Scien., 87 : 1069 (1998)) measures the IR spectrum in the amide 1 region and provides information on the secondary structure of the protein.
[0214] The iso-asp content in the samples is measured using the Isoquant Isoaspartate Detection System (Promega). The kit uses the enzyme Protein Isoaspartyl Methyltransferase (PIMT) to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the
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113/145 transfer of a methyl group from S-adenosyl-L-methionine to isoaspartic acid in the alpha-carboxyl position, generating S-adenosyl-L-homocysteine (SAH) in the process. This is a relatively small molecule and can usually be isolated and quantified by reverse phase HPLC using the SAH HPLC standards provided in the kit.
[0215] The potency or bioidentity of an antibody can be measured by its ability to bind to its antigen. The specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, for example, an immunoassay, such as the ELISA (enzyme-linked immunosorbent assay).
[0216] All publications mentioned in the present invention are incorporated by reference in order to describe and disclose methodologies and materials that can be used in connection with the present invention.
[0217] Having described different modalities of the invention in this document with reference to the accompanying drawings, it should be understood that the invention is not limited to these precise modalities and that various changes and modifications can be made by an element skilled in the art without departing from it. scope or spirit of the invention, as defined in the appended claims.
EXAMPLES
EXAMPLE 1
Anti-TIGIT Formulation Buffer Screening
[0218] A high yield formulation development study was carried out for three anti-TIGIT antibodies, each anti-TIGIT antibody with the following CDRs: HCDR1 from SEQ ID NO: 108, HCDR2 from SEQ ID NO: 154, HCDR3 from SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113, to assess (1) biophysical / biochemical liabilities; (2) conditions of
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114/145 pre-formulation (pH, salt and buffer) and (3) compatibility with the platform formulation. The samples were analyzed by UV / Vis spectrophotometry for turbidity (A350) as a substitute for larger aggregates, size exclusion chromatography (UP-SEC) to detect the formation of high molecular weight species, capillary isoelectric focus (clEF) to measure the effect of stress on the distribution of charges on the surface of the molecule, reducing capillary sodium dodecyl sulfate electrophoresis (CE-SDS) to detect proteolytic dividing of heavy or light chains, analysis of subvisible particles to detect subvisible aggregates.
[0219] The screening of the high-yield formulation consisted of 1 mg / mL of anti-TIGIT antibody formulated in a selection of three buffer species: acetate buffer with pH values ranging from 5.0 to 6.2, buffer citrate with pH values ranging from 5.6 to 6.8 and L-histidine buffer with pH values ranging from 5.0 to 6.8. Thus, pH values ranging from 5.0 to 6.8 and NaCI ionic strength from 0 to 150 mM were examined. The samples were stressed at 50 ° C for 10 days and analyzed for thermal stability by differential scanning fluorescence (DSF), colloidal stability by size exclusion chromatography (UP-SEC), propensity to aggregation using Guava (a characterization assay) subvisible based on flow cytometry), turbidity (A350 measurements), load variant profile (clEF) and fragmentation profile using Caliper.
[0220] Based on the results obtained from the study, the formulation that gave the protein maximum stability was 10 mM L-histidine in the pH range of 5.6 to 6.2. 10 mM L-Histidine in the pH range 5.6 to 6.2 showed minimal aggregation monitored by UP-SEC with main ASEC ranging from 1.63 to -1.85% (compared to main SEC ranging from -2 , 0 to -5.0% under other buffer and pH conditions) (data not shown). The clEF profile
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115/145 showed a decrease in the peak area of the main and basic species, and the increase in the peak area of the acid variants was observed in the samples after 10 days at 50 ° C (main clEF ranging from -9.0 to - 11 , 3%) (data not shown). A decrease in basic variants and an increase in acidic variants after exposure to elevated temperatures is a common occurrence for mAbs. The addition of salt reduced the stability of the protein in the studied compositions.
EXAMPLE 2
Study of pH range of Anti-TIGIT Formulation
[0221] In this study, an anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113, were tested at a concentration of 50 mg / ml in 10 mM Lhistidine buffer. 7% (w / v) sucrose was added to the formulation to increase the volume stability (as a stabilizer and non-ionic tonicity modifier) of the molecule. The anti-TIGIT antibody was formulated in 10 mM Lhistidine buffer, 7% sucrose at pH 5.5, pH 6.0 and pH 6.5. The stability of such formulations was assessed as follows:
(1) The stability of the molecule was monitored under accelerated thermal and storage stability conditions (5 ° C, 25 ° C and 40 ° C for up to 6 months), protected from light.
(2) Stability studies for freeze-thaw stress and agitation stress were also performed.
(3) A stirring study was carried out on formulations containing varying concentrations of polysorbate 80 (PS-80) to assess the concentration of PS-80 in the formulation.
(4) A mild stress study was conducted to assess the need for L-methionine in formulations.
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Materials and methods
Thermal stability study (3 months)
[0222] 50 mg / ml anti-TIGIT antibody was formulated in 10 mM Lhistidine buffer, 7% sucrose, 0.2 mg / ml polysorbate 80 at pH 5.5, pH 6.0 or pH 6 , 5. The resulting formulations were filtered sterile and filled in 2R flasks, covered with chlorobutyl stoppers and covered with aluminum caps with seals. The stability study was carried out at 5 ° C (ambient humidity), 25 ° C (60% relative humidity) and 40 ° C / (75% relative humidity). The samples were analyzed using UP-SEC, HP-IEX, clEF (selected samples), MFI, CESDS (non-reducing NR and reducing R), reducing peptide mapping (MFI and reducing peptide mapping was performed at selected points in time).
[0223] A 1-month thermal stability study was established for 25 mg / mL of anti-TIGIT antibody formulated in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 6.0. The resulting formulation was filtered sterile and filled with 2R flasks, capped with chlorobutyl stoppers and capped with aluminum seals. The stability study was carried out at 5 ° C (ambient humidity), 25 ° C (60% relative humidity) and 40 ° C / (75% relative humidity) for one month. The samples were analyzed using UP-SEC, clEF and CE-SDS (NR and R).
Stirring stability study
[0224] 50 mg / ml of anti-TIGIT antibody were formulated in 10 mM L-histidine buffer, 7% sucrose, pH 6.0 with various concentrations of polysorbate 80 (0, 0.1 and 0.2 mg / mL). The resulting formulations were filtered sterile and filled in 2R flasks (1.2 ml filling volume), capped with chlorobutyl stoppers and capped with aluminum caps with seals. The samples were shaken horizontally at 300 RPM for up to 7 days at 18 to 22 ° C. The samples were analyzed using UP-SEC, MFI, CE-SDS (NR
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117/145 and R).
Freeze and thaw stability
[0225] 50 mg / ml anti-TIGIT antibody was formulated in 10 mM Lhistidine buffer, 7% sucrose, 0.2 mg / ml polysorbate 80, pH 6.0. The resulting formulation was filtered sterile and filled with 2R flasks, covered with chlorobutyl stoppers and covered with aluminum caps with seals. The samples were subjected to 5 cycles of freezing and thawing from 80 ° C to 18 to 22 ° C (at least 24 hours in frozen conditions and at room temperature until complete thawing). The samples were analyzed using UP-SEC, MFI, CE-SDS (NR and R).
Study of the stability of light stress
[0226] Initial development studies indicated the presence of an exposed tryptophan residue, as well as some methionines that were responsible for oxidation under mild stress. The studies were carried out under conditions of visible light ICH stress (CWF, 0.1x ICH, 0.2x ICH, 0.5x ICH, 1x ICH) in formulations with and without L-methionine (Formulation 1: L- Histidine 1: 10 mM, 7% Sucrose, 0.2 mg / mL polysorbate 80, pH 6.0 and Formulation 2: 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, 7% sucrose, polysorbate 80 at 0.2 mg / ml, pH 6.0). The samples were analyzed using UP-SEC, clEF, CE-SDS (NR and R) and reducing peptide mapping.
Results
Thermal stability results
[0227] 50 mg / ml formulation: For all stability tests tested, no significant changes were observed at 5 ° C at all pH values after 3 months (data not shown). At 25 ° C and 40 ° C, pH 5.5 and pH 6.0 showed similar stability and these conditions were more stable than pH 6.5 (data not shown). Degradation rates at pH 6.5 for
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118/145 UP-SEC, CE-SDS (NR) and clEF assays at 25 ° C and 40 ° C were relatively higher than those observed at pH 5.5 and pH 6.0 and also when compared to the reference molecule. Therefore, a pH range of 5.5 to 6.0 (pl of 8.7) was considered adequate. No oxidation, deamidation or isomerization was observed for the anti-TIGIT antibody after 3 months at all temperatures and pH values.
[0228] Formulation of 25 mg / ml: Degradation was observed at 25 ° C and 40 ° C for all tests tested. The degradation rates were similar to the 50 mg / mL conditions (see above).
Stirring stability study
[0229] Formulations that did not contain polysorbate 80 showed visible particles at the end of 7 days. Invisible particle analysis showed that particles of 10 pm or more were significantly reduced in the formulation containing 0.2 mg / mL of polysorbate 80 concentration. No significant differences were observed between samples using the other assays.
Freeze and thaw stability
[0230] No changes in the stability of the molecule were observed after 5 cycles of freezing and thawing in all tests tested.
Study of the stability of light stress
[0231] Degradation was observed when samples from both formulations were exposed to light stress equal to or greater than 0.5x ICH when tested by UP-SEC, clEF, CE-SDS (NR and R). Conditions established below 0.5x ICH did not show significant degradation for both formulations. Reduced peptide mapping data under light stress conditions 0.5x and above showed oxidation of tryptophan and methionine residues. 10 mM L-methionine in the formulation reduced the levels of oxidation of methionine residues, but did not affect the levels of oxidation of tryptophan.
Conclusion
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[0232] Based on the above, 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, polysorbate 80 0.2 mg / mL pH 5.5-6.0, suitable to provide stability to withstand service life in refrigerated conditions.
EXAMPLE 3
Additional pH studies
[0233] An anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO : 112 and LCDR3 of SEQ ID NO: 113 and in an IgGl backbone was formulated in six 10 mM histidine buffers with different pH (ranging from 5.0 to 6.5). Thermal stability in different formulations was studied at 2 ~ 8 ° C, 25 ° C and 40 ° C over 8 weeks.
Formulation Anti-TIGIT antibody Plug PH Sucrose (% w / v) Polysorbate 80 (% w / v) 1 50 mg / ml 10 mM L-Histidine pH = 5.0 7% 0.02% 2 50 mg / ml 10 mM L-Histidine pH = 5.3 7% 0.02% 3 50 mg / ml 10 mM L-Histidine pH = 5.6 7% 0.02% 4 50 mg / ml 10 mM L-Histidine pH = 5.9 7% 0.02% 5 50 mg / ml 10 mM L-Histidine pH = 6.2 7% 0.02% 6 50 mg / ml 10 mM L-Histidine pH = 6.5 7% 0.02%
[0234] Histidine buffers of different pH (5.0 ~ 6.5) were prepared by titrating 10 mM L-histidine buffer in 10 mM Lhistidine-HCI buffer. The anti-TIGIT antibody was exchanged for buffer in six different histidine buffers with different pH through four to five cycles of ultrafiltration using the centrifugation device under the condition of 4 ° C and 4,500 rpm ~ 5,000 rpm (105 ~ 260 min in each round). After buffer exchange, the specific amount of sucrose and polysorbate 80 stock solution (1%, w / w) was added to different pH solutions to achieve the target amount and the appropriate amount of corresponding histidine buffer was added
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[0235] The formulations were then aseptically filtered with a 0.22 μηη membrane filter. 3 mL of each sample was aseptically filled in 6 mL glass vials for the TO thermal stability study, 4 weeks (4 W) and 8 weeks (8 W). 1 mL of each sample was aseptically filled in 6 mL glass flasks for the two-week thermal stability study (2 W). The filled vials were capped and sealed immediately after filling. All of the above steps were performed on the biosafety hood. These vials were placed in covered boxes and stored under different temperature conditions to study thermal stability.
Results and discussion
[0236] The appearance of all samples remained the same within four weeks in all conditions. However, after 8 weeks, samples at 2 ~ 8 ° C and 25 ° C showed a slightly yellow color and samples at 40 ° C showed a deeper yellow. All samples were slightly opalescent and free of visible particles during the study period. A considerable change in the protein concentration of all samples was not observed during the study.
[0237] The colloidal stability of the samples was assessed by size exclusion chromatography (SEC) for purity in which the percentage of monomer, the percentages of high molecular weight species (HMW) and the peak elution peaks (LMW species) . The analysis was performed using an Agilent 1260 Infinity system with the TSKGel G3000SWXL size exclusion chromatography column (300 χ 7.8 mm, 5 μπι). The mobile phase was 50 mM PB, 300 mM NaCI, pH 7.0 ± 0.2 and the flow rate was adjusted to 1.0 ml / min. The samples were diluted to 10 mg / mL for injection and detected at 280 nm with
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[0238] UPSEC data are shown in the table below:
2 to 8 ° C 25 ° C 40 ° CPH T0 2W 4W 8W 2W 4W 8W 2W 4W 8W % of Main Peak 5 97.7 97.5 97.4 97.5 97.7 97.4 97 97.2 96 94.5 5.3 97.7 97.5 97.7 97.4 97.6 97.2 96.8 96.5 96.2 94.6 5.6 97.6 97.4 97.6 97.3 97.1 97.1 96.8 97 96.1 94.8 5.9 97.5 97.3 97.5 97.3 97.3 97 96.5 96.2 96 94.7 6.2 97.3 97.2 97.5 97.1 97.1 96.8 96.5 96.4 95.7 94.3 6.5 97.3 97.1 97.4 97.1 96.9 96.6 96.2 95.8 95.5 94.6 % of HMW% of LMW 5 2.3 2.5 2.6 2.5 2.3 2.4 2.8 2.4 2.8 3.4 5.3 2.3 2.5 2.3 2.6 2.4 2.5 2.9 2.8 2.9 3.7 5.6 2.4 2.6 2.4 2.7 2.7 2.7 3 2.8 3.2 3.9 5.9 2.5 2.7 2.5 2.7 2.7 2.8 3.2 3.3 3.3 4.1 6.2 2.7 2.8 2.5 2.9 2.9 3 3.3 3.3 3.6 4.6 6.5 2.7 2.9 2.6 2.9 3.1 3.2 3.5 3.7 3.9 4.35 N, D, N, D „N, D, N, D, N, D 0.2 0.3 0.4 1.3 2.1 5.3 N, D, N, D, N, D, N, D, i N, D 0.2 0.2 0.6 0.8 1.7 5.6 N, D, N, D, N, D, N, D, 0.2 0.2 0.2 0.2 0.7 1.3 5.9 N, D, N, D, N, D, N, D, N, D 0.2 0.3 0.5 0.7 1.2 6.2 N, D, N, D, N, D, N, D, N, D 0.2 0.2 0.3 0.7 1.1 6.5 N, D, N, D, N, D, N, D, N, D 0.2 0.2 0.5 0.7 1.1
[0239] As shown in the table above, the main SEC% peak remained stable at 2 to 8 ° C in all samples; however, at 25 ° C and 40 ° C, a significant reduction in% of the main peak was observed. The rate of reduction in% of the main peak was faster in samples at 40 ° C than at 25 ° C. At 40 ° C for eight weeks, the% HMW was higher in the pH 6.2 and 6.5 samples, while the% LMW was higher in the pH 5.0 and 5.3 samples.
[0240] To assess the chemical stability of the formulations, capillary isoelectric focusing (clEF) was performed to assess chemical stability and monitor the change in the profile of the load variant over time. In summary, 20 μΙ (2.0 mg / mL) of the reference standard or the sample was mixed with 0.5 μΙ of marker pl 5.85, 0.5 μΙ of marker pl 9.77, 1 μΙ of Pharmalyte
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3-10, 0.5 μΙ Pharmalyte 5-8, 0.5 μΙ Pharmalyte 8-10.5, 35 μΙ 1% methyl cellulose, 37.5 μΙ urea 8 M. Purified water was added to compose a final volume of 100 ml. The mixture was then analyzed with the capillary isoelectric focus analyzer iCE-3 equipped with an entire column detection capillary coated with fluorocarbon. Focusing was carried out in two stages: (1) 1.5 kV for 1 min and (2) 3 kV for 8 min. During the experiment, the autosampler tray was maintained at 5 ° C.
[0241] The clEF data to assess the levels of acidic variants,% of peak main and% of basic variants are in the table below.
2 to 8 ° C 25 ° C 40 ° CPH T0 2W 4W 8W 2W 4W 8W 2W 4W 8W % of Main Peak 5 62.2 63 60 62.6 62.8 57.6 58.4 50.2 37.5 29.1 5.3 62.2 63 59.9 62.4 62.8 57.7 58.6 50.6 39.1 30.2 5.6 62.3 62.8 59.9 62.8 62.6 58.1 58.7 51.9 41.4 31 5.9 62 62.9 59.7 62.5 62.8 58.1 58.4 51.7 41.2 32.4 6.2 62.1 62.8 59.6 62.2 62.5 57.5 58.6 51.7 41.8 31.8 6.5 62.2 62.9 59.4 62.5 61.9 56.1 57.4 51.1 40.4 33.2 % acid 5 31.7 30.3 32.7 30.2 30.5 33.5 33.9 39.7 49.2 58.2 5.3 31.8 30.6 32.3 30.6 30.5 34 34.1 40.3 49.5 59.4 5.6 31.4 30.8 32.6 30.3 30.8 34.2 34.3 39.8 48.9 59.5 5.9 31.9 30.7 33.1 30.6 30.9 34.3 34.8 40.7 48.8 59.1 6.2 32.1 30.8 33.5 31.2 31.3 34.5 34.6 41 49 59.7 6.5 31.5 31 33.3 31 31.9 35.5 35.8 41.5 50.3 58.5 % of basic 5 6 6.7 7.3 7.2 6.8 8.9 7.7 10 13.3 12.6 5.3 6 6.4 7.7 7 6.7 8.3 7.3 9.1 11.3 10.3 5.6 6.2 6.5 7.5 6.8 6.5 7.6 7.2 8.2 9.6 9.5 5.9 6.1 6.4 7.3 6.9 6.3 7.5 6.7 7.6 9.9 8.4 6.2 5.9 6.4 6.9 6.6 6.3 8 6.8 7.2 9.3 8.6 6.5 6.3 6.2 7.2 6.4 6.2 8.4 6.8 7.3 9.4 8.3
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[0242] As can be seen in the table above, at 2 ~ 8 ° C, the% CIEF main peak,% acid peak and% basic peak was relatively stable and comparable across all samples.
[0243] At 2 ~ 8 ° C,% of the main basic peak of clEF was also increased; the increase in the pH 5.0 and 5.3 buffer samples was greater than in other samples. At 25 ° C, the main peak%, acid peak% and basic peak% remained stable for the first two weeks, but changed slightly after four weeks, where the main peak% decreased while the acid peak% increased correspondingly . The rate of change in different formulations was comparable. At 40 ° C, a significant reduction in the% of the main peak and a notable increase in the% of the peak acid were found in all formulations, even after two weeks, but the extent of the change was similar in each formulation. The basic% of peak was also increased; the increase in the pH 5.0 and 5.3 buffer samples was greater than in other samples.
[0244] To evaluate the purity of the formulations, a non-reducing analysis of Caliper was performed. In summary, the sample buffer purchased was mixed with a 10% solution of sodium dodecyl sulfate (SDS) in a 20: 1 volume ratio, and a 100 mM N-ethylmaleimide solution was added to the mixed solution in a volume ratio. 0.7 to 20 (referring to the sample denaturation solution). The standard or sample was diluted to 1 mg / mL first and 2 μΙ of diluted standard or sample was mixed with 7 μΙ of sample denaturing solution. The mixture was incubated at 70 ° C for 10 min. 35 μΙ of purified water was added to the incubated solution and 42 μΙ of the mixed solution was transferred to a 96-well plate for analysis. The sample plate was analyzed with the LabChip GX II HT, using the HT Antibody Analysis 200 assay.
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[0245] The data from the non-reducing Caliper analysis to assess the% purity are shown in the table below:
2 to 8 ° C 25 ° C 40 ° CpH TO 2 W 4 W 8 W 2 W 4 W 8 W 2 W 4 W 8 W Non-reducing caliper % Purity 5 96 95.2 94.6 94.9 94.9 93.6 94.1 91.4 87.8 84.4 5.3 96 95 94.7 94.9 94.8 93.8 94.1 92.7 89.5 86.4 5.6 96.1 95 94.6 94.9 94.8 93.9 94.1 93 90.7 88.5 5.9 96.3 94.9 94.5 94.9 94.8 93.9 94.2 93.4 91.1 89.3 6.2 96.2 94.9 94.5 95 94.8 93.7 94.5 93.3 91.3 88.7 6.5 96 95.1 94.4 94.9 94.7 93.4 94.1 93.2 90.7 89.3
[0246] As shown in the table above at 2 ~ 8 ° C for eight weeks, the reducing purity with Caliper_Non in each formulation was relatively stable. At 25 ° C for eight weeks, the purity in each formulation decreased slightly. At 40 ° C, the purity decreased significantly, especially when the samples were in pH 5.0 and 5.3 buffer, the decrease was much faster than that of others. The molecular size was stable during the study (data not shown).
[0247] To further evaluate the purity of the formulations, analysis of reducing caliper was also performed. In summary, the acquired sample buffer was mixed with 10% SDS solution in a 20: 1 volume ratio and 1M dithiothreitol solution was added to the mixed solution in a 0.7 to 20 volume ratio (referred to the denaturation of the sample). The standard or sample was diluted to 1 mg / mL first and 2 μΙ of diluted standard or sample was mixed with 7 μΙ of sample denaturing solution. The mixture was incubated at 70 ° C for 10 min. 35 μΙ of purified water was added to the incubated solution and 42 μΙ of the mixed solution was transferred to a 96-well plate for analysis. The sample plate was analyzed with the LabChip GX II HT, using the HT Antibody Analysis 200 assay.
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[0248] The analysis data of the reducing caliper to evaluate the% purity are shown in the table below:
2 to 8 ° C 25 ° C 40 ° C pH T0 2W 4W 8W 2W 4W 8W 2W 4W 8W % Purity (CaliperR) 5 99.4 99.4 99.3 99.2 99.3 99.1 98.9 98 97.1 91.3 5.3 99.4 99.4 99.2 99.2 99.3 99 99 98.5 97.7 94.9 5.6 99.4 99.4 99.3 99.1 99.3 99.2 98.8 98.7 98.2 96.8 5.9 99.4 99.4 99.2 99.1 99.2 99.2 98.8 99.1 98.6 96.8 6.2 99.3 99.3 99.3 99.2 99.3 99.2 98.9 98.9 98.7 96.9 6.5 99.3 99.4 99.2 99.1 99.3 99.2 98.8 98.9 98.5 97.5
[0249] As seen above, at 2 ~ 8 ° C and 25 ° C for eight weeks, the CaliperReduced purity in each formulation was relatively stable. At 40 ° C, an apparent decline in the purity of Caliper_R was found in all formulations. The purity of the samples in the pH 5.0 buffer had the greatest decrease, followed by the samples in the pH 5.3 buffer. The rate of decrease in buffers of pH 5.6, 5.9 and 6.2 was comparable, but slower. The purity of the samples in the pH 6.5 buffer had the slowest decrease. The decline in purity probably occurred due to the decrease in the% heavy chain (HC) while the% DA light chain (LC) was stable in the study. The antibody light chain and the size of the heavy chain were stable in all samples above 8 S.
EXAMPLE 4
Anti-TIGIT formulation without methionine
[0250] An anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO : 112 and the LCDR3 of SEQ ID NO: 113, was tested at a concentration of 50 mg / mL in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80 with pH ranging from 5 , 0 to 6.5. The stability of the molecule was monitored under accelerated thermal and storage stability conditions, protected from light. In addition to thermal stability,
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Results
Thermal stability study (8 weeks)
[0251] For all stability indication assays tested, the anti-TIGIT antibody was stable at 5 ° C for all tested responsibilities. The degradation rates observed using UP-SEC, Caliper CE-SDS, clEF, MFI were higher at 40 ° C than at 25 ° C. At 25 ° C and 40 ° C, the following results were remarkable:
[0252] UP-SEC: A decline in monomer% was observed for all pH values from 5.0 to 6.5. At the lowest pH values (5.0 and 5.3), the main peak decline occurred mainly due to the increase in% of low molecular weight species (LMW), while the% decline in monomers at pH 6.5 occurred mainly due to the increase in% of species of high molecular weight (HMW). The% monomer decline was higher at pH 6.5 after 8 weeks of accelerated stability, (data not shown)
[0253] clEF: the decline in the main peak of clEF was observed for all pH values from 5.0 to 6.5 at 25 ° C and 40 ° C. At the highest pH values (6.3-6.5), the main decline in the peak occurred mainly due to the increase in acidic variants, while at pH 5.0 and 5.3, the main decline in the peak occurred due to the increase in acidic and basic variants, (data not shown)
[0254] CE-SDS (Caliper): The main decline in the non-reducing CE-SDS peak was observed mainly at 40 ° C due to the presence of fragmented species. Formulations with lower pH values (5.0 and 5.3) showed a relatively higher fragmentation rate than the rest
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[0255] The reducing analysis of CE-SDS (Caliper) revealed that at 5 ° C and 25 ° C for eight weeks, the purity in each formulation was relatively stable. At 40 ° C, an apparent decline in purity was found in all formulations. The purity of the samples in the pH 5.0 buffer had the greatest decrease, followed by the samples in the pH 5.3 buffer.
[0256] MFI: Subvisible particle increases were observed for all formulations at 40 ° C. PH 6.3 and 6.5 had the highest increases in subvisible particles over the rest of the buffers.
[0257] Reducing peptide mapping: Among the identified disadvantages, only M254 showed a relative increase in oxidation after 8 weeks at 40 ° C compared to the initial samples.
[0258] Conclusion: 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 5.6-6.3 adequately supported the storage stability of the anti-TIGIT antibody for 8 weeks.
Stirring stability study
[0259] No changes were observed in soluble aggregates, charged variants, fragmentation or invisible particles when the 50 mg / mL antiTIGIT formulation (in 10 mM L-histidine buffer, 7% sucrose, pH 5.8 and 0. 0 , 1, 0.2 and 0.3 mg / mL PS-80) was slightly stirred for up to 7 days at 100 RPM at 18 to 22 ° C.
Freeze and thaw stability
[0260] No changes were seen in soluble aggregates, charged variants, fragmentation or invisible particles when 50 mg / mL of anti-TIGIT antibody (in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 5.8) after 5 freeze / thaw cycles (frozen at -80 ° C for 2 hours and thawed at room temperature for 1 hour)
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Study of the stability of light stress
[0261] The 50 mg / mL formulation was subjected to 48 hours under visible light stress (5,000 Ix). Under these conditions, there was a minimal change in soluble aggregates, charged variants, invisible particles, pH, concentration, fragmentation and oxidation under conditions of ~ 0.2x ICH (12H light exposure). Under lx conditions (exposure to 48H light), there was an increase in soluble aggregates, acidic variants, fragmentation, subvisible particles and methionine oxidation.
Conclusion
[0262] Based on these studies, 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 5.3-6.3 was able to support antibody stability anti-TIGIT. Methionine oxidation was observed after exposure to mild intense stress. As noted in Example 2, the addition of 10 mM L-methionine reduces the oxidation of methionine residues.
EXAMPLE 5
Polysorbate Screening 80
[0263] An anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO : 112 and the LCDR3 of SEQ ID NO: 113, was formulated in four (4) formulations of pH 5.8, 10 mM L-histidine buffer and with different concentrations of PS-80 (as shown below). The stability of the protein in different formulations was studied with or without agitation for a period of 7 days at 20 ° C.
Formulation Anti-TIGIT antibody Plug P Sucrose (% w / v) Polysorbate 80 (mg / mL) 1 50 mg / ml 10 mM L-Histidine pH = 5.8 7% 0 mg / ml
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2 50 mg / ml 10 mM L-Histidine pH = 5.8 7% 0.1 mg / ml 3 50 mg / ml 10 mM L-Histidine pH = 5.8 7% 0.2 mg / ml 4 50 mg / ml 10 mM L-Histidine pH = 5.8 7% 0.3 mg / ml
[0264] The formulations were formulated in 10 mM Lhistidine buffer at pH 5.8 using a laboratory-scale TFF buffer exchange system. The proteins formulated with different levels of polysorbate 80 were then aseptically filtered with a 0.22 µm membrane filter. 2 ml of each sample was then aseptically filled in 6 ml glass vials. The filled vials were capped and sealed immediately after filling. The samples were divided into a shaking group and a non-shaking group. In the stirring group, these vials were transferred to covered boxes and then placed on the thermostat shaker and stirred at 100 rpm, 20 ° C for up to 7 days. In the no-shake group, these vials were transferred to covered boxes and placed on the thermostat stirrer, but the stirrer was kept at 20 ° C for up to 7 days.
[0265] The stability of the antibody in the different formulations with or without agitation was studied after 3 and 7 days.
[0266] UPSEC data to assess levels of High Molecular Weight Species (HMW or aggregates),% monomer and LMW (Low Molecular Weight species) are in the Table below:
3 days 7 daysFormulation T0 No shaking 100 rpm No shaking 100 rpm % of Main Peak 1 97.1 97.2 97.2 97.1 97.2 2 97 97.1 97.1 97 97 3 97 97.1 97.1 96.9 96.9 4 97.1 97.1 97.1 96.9 96.9 % Molecular Weight 1 2.9 2.8 2.8 2.9 2.8 2 3 2.9 2.9 3 3
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High (HMW) 3 3 2.9 2.9 3.1 3.1 4 2.9 2.9 2.9 3.1 3.1 Low Molecular Weight% (LMW) 1 N.D. N.D. N.D. N.D. N.D. 2 N.D. N.D. N.D. N.D. N.D. 3 N.D. N.D. N.D. N.D. N.D. 4 N.D. N.D. N.D. N.D. N.D.
[0267] As can be seen, the polysorbate 80 content did not have a significant impact on the purity of the SEC in the condition of with or without agitation. The polysorbate 80 content did not significantly impact the pl, the percentage of main peak, acid peak and basic peak in the clEF assay with or without agitation for up to 7 days.
3 days 7 days T0 No shaking 100 rpm No shaking 100 rpm % inMain Peak 1 61.5 61.4 61.4 62.4 62.3 2 61.5 61.5 61.9 61.9 62.1 3 61.2 60.8 60.5 61.5 61.6 4 61.9 61.4 61.5 62 61.8 % acid 1 30.9 31.1 30.5 31.1 30.6 2 31.2 31.6 30.5 31.3 31.1 3 31.5 32.2 32.3 31.5 31.6 4 31.1 31.8 31.4 31.4 31.5 % of basic 1 7.6 7.5 8.1 6.5 7.1 2 7.3 6.9 7.6 6.8 6.7 3 7.3 7 7.2 6.9 6.8 4 7 6.7 7 6.7 6.7
[0268] Polysorbate 80 content had no significant impact on purity with Non-Reducing Caliper with or without stirring for up to 7 days, as shown in the Table below.
Formulation T0 3 days 7 days No shaking 100 rpm No shaking 100 rpm Non-Reducing Caliper
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% Purity 1 94.9 94.8 95 94.4 94.5 2 94.9 94.8 94.6 94.7 94.4 3 94.7 94.8 94.8 94.3 94.4 4 94.9 94.8 94.6 94.3 94.3
[0269] Caliper analysis was also performed. The PS-80 content had no significant impact on Caliper_Redutor purity with or without agitation in 7 days.
3 days 7 daysFormulation T0 No shaking 100 rpm No shaking 100 rpm Non-Reducing Caliper % Purity 1 99.2 99.2 99.2 99.2 99.2 2 99.2 99.3 99.2 99.2 99.2 3 99.1 99 99.2 99.2 99.2 4 99.2 99.1 99.2 99.2 99.1
[0270] In order to measure invisible particles, approximately 1,500 μΙ of each sample was taken from the glass vial container and tested by microflow imaging (MFI) according to the user manual. The concentration of particles in different size ranges, including 1 ~ 2 pm, 2 ~ 5 pm, 5 ~ 10 pm, 10 ~ 25 pm and> 25 pm have been reported (see below). The polysorbate 80 content had no significant impact on the concentration of particles with or without agitation for up to 7 days.
3 days 7 daysFormulation T0 No shaking 100 rpm No shaking 100 rpm 1 pm <ECD <2 pm 1 8318 AT 9080 1205 2046 2 25396 10831 4429 7668 5898 3 8048 2660 15877 2952 4932 4 8867 2590 13115 9214 2728 2 pm <ECD < 1 1792 4516 2700 140 574 2 6068 4215 711 1137 806
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3 days 7 daysFormulation T0 No shaking 100 rpm No shaking 100 rpm 5 pm 3 818 363 3950 927 735 4 1321 297 3476 2418 310 5 pm <ECD <10 pm 1 369 AT 486 27 52 2 770 647 79 237 63 63 3 121 63 379 121 94 4 104 80 253 167 61 10 pm <ECD <25 pm 1 78 AT 103 2 22 2 135 73 7 59 5 3 39 13 66 20 8 4 14 25 34 16 8 ECD>25 pm 1 0 AT 6 0 2 2 7 14 3 21 15 3 10 10 10 4 2 6 12 0 12
EXAMPLE 6
Addition of chelator
[0271] This study compared the stability of an anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111 , LCDR2 of SEQ ID NO: 112 and LCDR3 of SEQ ID NO: 113 in 10 mM L-histidine buffer (pH = 5.8), polysorbate 0.02% (w / v) 80, L-methionine at 10 mM (L-Met), 7% w / v sucrose in the presence or absence of 20 µM or 50 µM DTPA.
[0272] The three formulations were placed in bottles and placed in stability at 5 ° C (ambient humidity), 25 ° C (60% relative humidity) and 40 ° C (75% relative humidity) for eighteen weeks protected from light .
Formulation Antibody LHistidine Cap Sucrose L-Met Polysorbate anti-TIGIT (pH % (w / v)80% (w / v) 5.8)
DTPA
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1 50 mg / ml 10 mM 7% 10 mM 0.02% 0 2 50 mg / ml 10 mM 7% 10 mM 0.02% 20 µM DTPA 3 50 mg / ml 10 mM 7% 10 mM 0.02% 50 µM DTPA
[0273] The colloidal stability of the samples was evaluated by size exclusion chromatography (SEC) for the purity in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and peak elution peaks ( LMW species). UPSEC data to assess the levels of% HMW (aggregates),% monomer and%
LMW are in the table below:
5 ° C 25 ° C 40 ° CForm T0 4W 8W 18W 4W 8W 18W 2W 4W 8W 18W %inHMW 1 1.54 AT 1.57 1.57 1.53 1.57 1.64 1.51 1.61 1.72 2.2 2 1.57 1.59 1.59 1.57 1.56 1.57 1.59 1.51 1.56 1.65 1.94 3 1.61 1.61 1.61 1.58 1.56 1.57 1.59 1.53 1.57 1.63 1.91 % Monomer 1 98.2 AT 98.2 98.2 98.1 98.0 97.8 97.9 97.6 97.2 95.7 2 98.2 98.1 98.1 98.2 98.1 98.0 97.9 98.0 97.7 97.3 96.1 3 98.1 98.1 98.1 98.2 98.1 98.0 97.9 98.0 97.7 97.4 96.2 %inLMW 1 0.30 AT 0.28 0.22 0.36 0.42 0.54 0.55 0.82 1.11 2.07 2 0.28 0.28 0.27 0.22 0.35 0.40 0.55 0.53 0.77 1.02 1.94 3 0.25 0.27 0.26 0.23 0.35 0.41 0.56 0.51 0.77 1.02 1.92
[0274] As shown in the table above, at 5 ° C, 25 ° C and 40 ° C, all three formulations showed an increasing trend in% HMW peak and% LMW peak (and a consequent decrease in% peak monomer) for up to a point in time of 18 weeks. At 25 ° C, both formulations showed similar trends, but minor changes, compared to 40 ° C. At 5 ° C, no substantial changes were observed. Formulation 1 shows a greater increase in% HMW and% LMW compared to Formulation 2 (20 µM DTPA) and Formulation 3 (50 µM DTPA). In addition, Formulation 1 showed a greater decrease in the% monomer in
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[0275] To assess whether DTPA can protect formulations from oxidative stress, the three formulations were placed in bottles and exposed to light (0.5X ICH and IX ICH). As seen in the table below, Formulation 1 shows a greater increase in the% oxidation of M254, M430 and W104 (the methionines and tryptophan which are susceptible to oxidation) compared to Formulation 2 (20 µM DTPA) and Formulation 3 (DTPA one). Thus, DTPA can further improve the stability of the anti-TIGIT antibody formulation.
Formulation1 Formulation 1 Formulation 2 Formulation 3Dark Control 0.5X ICH IX ICH 0.5X ICH IX ICH 0.5X ICH IX ICH LC_M4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 HC_M34 0.3 0.3 0.3 0.3 0.3 0.3 0.4 HC_M81 0.2 0.2 0.2 0.2 0.2 0.2 0.3 HC_M254 3.6 16.3 30.2 13.7 27.5 15.9 21.5 HC_M430 1 10.9 19.2 9.3 18.3 8.6 16.2 HC_W104 0.6 8.8 17.7 7.3 16.7 6.9 12.1
tn0η 25 ° C 40 ° CForm TO 4 8W 18W 4 W 8 W 18W 2 4 8 18 Cone, from PS 80 1 0.24 0.23 0.23 0.22 0.20 0.18 0.16 0.19 0.17 0.16 0.13 2 0.24 0.23 0.23 0.21 0.20 0.18 0.16 0.19 0.16 0.16 0.14 3 0.22 0.22 0.22 0.21 0.19 0.17 0.15 0.18 0.16 0.15 0.13
EXAMPLE 7
Long-term stability of anti-TIGIT antibody formulations
[0276] This example describes long-term stability data for an anti-TIGIT antibody formulated in a buffer of L-histidine, L-methionine, sucrose, polysorbate 80 and water for injection, as follows:
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Ingredients Concentration (mg / mL) Amount (mg / vial) Active Anti-antibodyTIGIT 50.0 100.0 Inactive (Excipients) L-Histidine 0.465 0.930 L-HistidineMonohydrochloride monohydrate 1.47 2.94 L-Methionine 1.49 2.98 Sucrose 70 140.0 Polysorbate 80 0.20 0.40 Water for Injection AT Enough quantity for 2.0 ml
[0277] The solutions were filled in a USP Type 1 glass bottle with an elastomeric stopper and aluminum seal. The flasks were then incubated in three different storage conditions: 5 ° C (ambient humidity), 25 ° C (60% relative humidity) and 40 ° C (75% relative humidity). Data are collected at zero time, 1 month, 3 months, 6 months for all storage conditions, 9 months (storage conditions of 5 ° C and 25 ° C), 12 months (storage conditions of 5 ° C and 25 ° C), 18 months (storage conditions at 5 ° C), 24 months (storage conditions at 5 ° C) and 36 months (conditions at 5 ° C).
Results
[0278] The results demonstrate general physical and chemical stability of the anti-TIGIT antibody when stored under the recommended long-term conditions of 5 ° C for 18 months. There was no measurable loss of power observed and the purity was within specifications under the recommended storage conditions. The results are presented in the following tables:
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5 ° C / Ambient humidity Measured Attribute Point in Time (months)Initial 1 3 6 9 12 18 Biological Potency byELISA ofLink 96 92 100 94 101 92 99 Purity per UPSEC%Kind ofHigh WeightMolecular (%) 1.33 1.48 1.40 1.63 1.65 1.66 1.77 Monomer (%) 98.7 98.5 98.6 98.3 98.3 98.3 * Kind ofLow weightMolecular (%) <QL <QL <QL <QL <QL <QL <QL Load Variants by% of HP-IEXAcid variants 21.46 21.54 22.04 22.51 22.49 22.48 22.92 Total principal 68.8 68.4 67.9 67.3 67.1 67.3 67.1 Basic variants 9.70 10.11 10.06 10.15 10.38 10.20 9.95 Purity by% of non-reducing CE-SDS 96.4 96.4 96.3 96.2 96.0 95.9 95.7 Purity by% of CE-SDS Reducer 98.1 98.2 98.0 98.1 98.0 98.1 97.7 pH 6.1 6.1 6.1 6.0 6.0 6.0 6.0 Protein Concentration 51.7 51.4 51.2 51.0 51.5 50.9 51.5 UV A350 0.150 0.142 0.138 0.144 0.146 0.145 0.150 QL = Limit of Quantification (0.4%) * value is 98.2 25 ° C / 60% Relative Humidity Attribute Point in Time (months)
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Measured Initial 1 3 6 9 12 Biological Potency byELISA ofLink 96 90 94 94 98 95 Purity per UPSEC%Kind ofHigh WeightMolecular (%) 1.33 1.57 1.48 1.72 1.77 1.83 Monomer (%) 98.7 98.4 98.3 97.9 97.7 97.4 Kind ofLow weightMolecular (%) <QL <QL <QL <QL <QL <QL Load Variants by% of HP-IEXAcid variants 21.46 23.19 28.74 34.16 39.86 44.04 Total principal 68.8 65.8 59.7 53.8 47.6 43.5 Basic variants 9.70 11.06 11.51 12.02 12.55 12.44 Purity by% of non-reducing CE-SDS 96.4 96.0 95.2 94.4 93.2 92.1 Purity by% of CE-SDS Reducer 98.1 98.0 97.8 97.4 96.7 96.1 pH 6.1 6.1 6.1 6.0 6.0 6.0 Protein Concentration 51.7 51.1 51.1 50.9 51.5 50.9 UV A350 0.150 0.150 0.163 0.179 0.196 0.209 QL = Limit of Quantification (0.4%)
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40 ° C / 75% Relative Humidity Measured Attribute Point in Time (months)Initial 1 3 6 9 12 Biological Potency byELISA ofLink 96 96 99 94 ND ND Purity per UPSEC%Kind ofHigh WeightMolecular (%) 1.33 1.64 1.78 2.63 ND ND Monomer (%) 98.7 97.9 96.9 93.8 ND ND Kind ofLow weightMolecular (%) <QL <QL <QL <QL <QL <QL Load Variants by% of HP-IEXAcid variants 21.46 38.03 61.62 80.02 ND ND Total principal 68.8 47.8 25.1 10.0 ND ND Basic variants 9.70 14.14 13.29 9.95 ND ND Purity by% of non-reducing CE-SDS 96.4 93.6 88.9 79.6 ND ND Purity by% of CE-SDS Reducer 98.1 96.9 94.2 87.7 ND ND pH 6.1 6.1 6.1 6.0 ND ND Protein Concentration 51.7 52.0 51.2 51.4 ND ND UV A350 0.150 0.188 0.251 0.453 ND ND QL = Limit of Quantification (0.10%)
Protein concentration
[0279] Protein concentration stability data for all
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139/145 the moments and conditions did not change significantly due to the time or condition of storage and all results were within the acceptance criteria of 45 to 55 mg / mL.
J3H
[0280] There was no significant change in pH for the conditions of 5 ° C, 25 ° C and 40 ° C. Figure 1 shows the pH data for the period from 0 to 9 months.
Polysorbate 80
[0281] Polysorbate 80 content at the recommended storage condition of 5 ° C decreased slightly to 0.13 mg / mL at 9 months and 18 months (18 month data not shown). A decreasing trend was observed in polysorbate 80 to 25 ° C (accelerated) and 40 ° C (stressed). At 40 ° C, the concentration of polysorbate 80 decreased to 0.06 mg / mL in 6 months and the content of polysorbate 80 at 25 ° C decreased to 0.07 mg / mL in 9 months. Data on polysorbate 80 concentration for up to 9 months are shown in Figure 2.
Power connection by ELISA
[0282] There was no evident trend at any time or condition in the ELISA results obtained. Power data for up to 9 months is shown in Figure 3.
Purity by UP-SEC
[0283] UP-SEC purity data are illustrated below in Figure 4 for% monomer, Figure 5 for% high molecular weight species and Figure 6 for% low molecular weight species, up to 9 months.
[0284] Under the recommended storage conditions of 5 ° C, there is a slight decrease in the% monomer with a slight corresponding increase in the% of high molecular weight species over 18 months of stability. The% of low molecular weight species from the beginning to 18 months is below the limit of quantification (<QL), which is equal to 0.4%. At 25 ° C, the%
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[0285] In the stressed condition of 40 ° C, the% monomer decreased from 98.7% to 93.8%, with corresponding increases in high molecular weight species from 1.33% to 2.63% and in species of low molecular weight from <QL to 3.53%. This result was not unexpected, given the nature of the storage condition.
Reducer and non-reducer CD-SDS
[0286] Figures 7 and 8 show the purity data for up to 9 months, as determined by reducing and non-reducing CD-SDS. There were no notable trends in the reducing (% heavy and light chain) or non-reducing (% intact IgG) CE-SDS in the 5 ° C long-term storage condition and the results were within the GMP acceptance criteria of> 90.0%. In the accelerated condition of 25 ° C, a decreasing trend was observed for the non-reducing condition. For the reducing CE-SDS condition, a decreasing trend was also observed. For the reducing and non-reducing CE-SDS, all results up to the nine-month period were within the GMP acceptance criteria. In the stressed condition at 40 ° C, in 6 months, the results of reducing and non-reducing CD-SDS were below the 90.0% acceptance criteria defined for the GMP drug. The result of the non-reducing CE-SDS went out of specification in 3 months, with a result of 88.9% and then decreased further in 6 months, to 79.6%. For the reducing CE-SDS, the% of heavy chain and% of light chain decreased from 94.2% in 3 months to 87.7% in 6 months. This reduction was not unexpected at 40 ° C, considering the nature of the condition.
Load variants by HP-IEX
[0287] At 5 ° C (long-term storage), there is a slight increase in%
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141/145 of acid variants from onset at 21.46% to 9 months at 22.49%, with a corresponding slight decrease in the grand total from 68.8% to 67.1% at 9 months. The% of basic variants starts to increase slightly at 9 months, from 10.15% at 6 months to 10.38% at 9 months. At 25 ° C (accelerated), the Grand Total fell from 68.8% in the initial moment to 47.6% in 9 months. Along with a reduction in the Main Total, there was a corresponding increase in Acid Variants from 21.46% to 39.86% and a slight increase in Basic Variants from 9.70% to 11.51%. At 40 ° C (stressed), there was a considerable decrease in the Grand Total to 10.1% in 6 months, together with a corresponding corresponding increase in the Acid Variants to 80.02% and in the Basic Variants to 9.95%.
Particulate material
[0288] The particulate material was measured by mHIAC. Results at 5 ° C were well below the acceptance criteria of <6,000 particles per container for> 10 pm and <600 particles per container for> 25 pm from baseline to 9 months. At 25 ° C, an increase in particles> = 10 pm has been reported from 13 particles per container at the start to 460 particles per container in 9 months. There was a decrease in particles for particulates> = 25 pm with a result of 3 particles per container at 9 months. All time points for data at 25 ° C were within the acceptance criteria for the analyzes> = 10 pm and> = 25 pm. Data at 40 ° C showed a dramatic increase in particles> = 10 pm with 8,258 particles per container at 9 months. This result was outside the acceptance criteria of <= 600 particles per container. The result for particles> = 25 pm increased in the stability period of 9 months to 124 particles per container, meeting the acceptance criteria> = 25 pm (<= 600 particles per container).
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Turbidity
[0289] Turbidity was determined from spectrophotometric absorbance at 350 nm. In the condition of long-term storage at 5 ° C, there were no noticeable changes until the 9-month period. At 25 ° C, there is a slight increase in 3 months with a result of 0.163 AU and continues to increase to 9 months with a result of 0.196 AU. At 40 ° C, there is a more pronounced increase from 1 month with 0.188 AU and then increasing considerably to 0.453 AU at 9 months.
Conclusions
[0290] Based on the data, at the 18-month test date, no major changes or trends were observed in the 5 ° C storage condition throughout the stability studies for pH, protein concentration, appearance and visible particles (data not shown) and power and particulate matter (data not shown). With the exception of a slight increase in color and a decrease in PS-80 content to 0.13 mg / mL, no noticeable changes or trends were observed for any stability test at 5 ° C.
[0291] Based on the data for long-term stability of 5 ° C, the anti-TIGIT formulation containing L-histidine buffer, sucrose, polysorbate 80 and L-methionine has an expected shelf life of 30 months. Formulations that further comprise a chelator are expected to reduce the degradation of polysorbate 80 that has been observed.
EXAMPLE 8
Co-formulation of an anti-TIGIT antibody and an anti-PD-1 antibody.
[0292] Co-formulation of two antibodies in a single formulation is more convenient for patients and increases compliance with the dosage of the two antibodies together. The co-formulation of two antibodies in a single
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143/145 formulation is more convenient for patients and increases compliance with the dosage of the two antibodies together. An anti-TIGIT antibody with the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113 in an IgGl backbone was co-formulated with pembrolizumab. Based on protein-protein interactions (shown below), co-formulation (shown below) was stable at pH 5.0-6.0. Therefore, the co-formulation (P1T1) at pH 5.0, 5.5 and 6.0 was chosen and placed in additional thermal stability at 5 ° C, 25 ° C and 40 ° C, together with the two controls (antibody PD1 and anti-TIGIT antibody).
Coformula-tions ReasonPembrolizumab /Anti-TIGIT AntibodyÍEZel Pembrolizumab AntibodyAnti-TIGIT Total Concentration P1T1 1: 1 20 mg / ml 20 mg / ml 40 mg / ml PI (Control) 1: 0 20 mg / ml none 20 mg / ml IT (Control) 0: 1 none 20 mg / ml 10 mg / ml
[0293] The formulations were prepared as liquid formulations as follows:
Formulation Plug PH Cryoprotectant / Tonicity Modifier Tensoact 0 Antioxidant P1T1 L-Histidine(10 mM) 5,5.5,6 Sucrose (7%) PS-80(0.02%) 10 mM ofL-Met PI L- 5, Sucrose (7%) PS-80 10 mM of
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Formulation Plug PH Cryoprotectant / Tonicity Modifier Tensoact 0 Antioxidant (Control) Histidine(10 mM) 5.5,6(0.02%) L-Met IT (Control) L-Histidine(10 mM) 5,5.5,6 Sucrose (7%) PS-80(0.02%) 10 mM ofL-Met
[0294] Each formulation was filled to 1 mL in 2R flasks. Stability will be measured by visual inspection, protein concentration, microflow imaging (MFI) (particulate evaluation), mixed mode size exclusion chromatography (SEC) (aggregation evaluation, IEX (load variant evaluation) and UP -SEC (aggregation assessment). The thermal stability protocol is as follows:
T0 1 month 2 months 3 months Five months Extra 5 ° C (Ambient humidity) 1 combo+ 2 mono 1 combo+ 2 mono 1 combo + 2 mono 1 combo + 2 mono 1 combo+ 2 mono 1 combo+ 2 mono 25 ° C /60% ofMoistureRelative1 combo+ 2 mono 1 combo + 2 mono 1 combo + 2 mono 1 combo+ 2 mono 1 combo+ 2 mono 40 ° C /75% Relative Humidity1 combo+ 2 mono 1 combo + 2 mono 1 combo + 2 mono 1 combo+ 2 mono 1 combo+ 2 mono
[0295] Protein-protein interactions, which are indicative of the colloidal and thermal stability of different coformulations, were measured. A repulsive protein-protein interaction, as indicated by a positive diffusion interaction parameter (Kd) value of Kd> 0, indicates a stable formulation with a low propensity for aggregation. It was found that the Kd for co-formulation had a positive Kd value, which is indicative of repulsive protein-protein interaction
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145/145 and stabilizer, which would indicate less propensity to aggregate and stable co-formulation.
[0296] Based on the positive diffusion interaction parameter (Kd) or Kd> 0, antibodies when co-formulated are expected to behave well when co-formulated together, similar to single antibody formulations.

权利要求:
Claims (30)
[1]
1. Formulation, characterized by the fact that it comprises:
(i) about 10 mg / ml to about 200 mg / ml of an anti-TIGIT antibody, or antigen-binding fragment thereof;
(ii) about 5 mM to about 20 mM buffer;
(iii) about 6% to about 8% weight / volume (w / v) of non-reducing sugar;
(iv) about 0.01% to about 0.10% (w / v) of non-ionic surfactant; and (v) about 1 mM to about 20 mM of antioxidant.
[2]
Formulation according to claim 1, characterized in that the anti-TIGIT antibody or antigen-binding fragment thereof comprises three light chain CDRs comprising CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: 112 , CDRL3 of SEQ ID NO: 113 and three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154 and CDRH3 of SEQ ID NO: 110.
[3]
Formulation according to claim 1 or 2, characterized in that the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 148 and a variable chain region light comprising SEQ ID NO: 152.
[4]
Formulation according to claim 3, characterized in that the anti-TIGIT antibody comprises: (i) a human IgG 1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291 and a constant domain human kappa light chain comprising the amino acid sequence of SEQ ID NO: 293; or (ii) a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293.
[5]
Formulation according to any one of claims 1 to 4,
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2/5 characterized by the fact that it has a pH between 5.3 and 6.2.
[6]
Formulation according to any one of claims 1 to 5, characterized in that the buffer is a L-histidine buffer, the non-reducing sugar is sucrose, the non-ionic surfactant is polysorbate 80 and the antioxidant is L -methionine, the formulation comprising:
(i) about 10 mg / ml to about 200 mg / ml of an anti-TIGIT antibody, or antigen-binding fragment thereof;
(ii) about 5 mM to about 20 mM of an L-histidine buffer;
(iii) about 6% to about 8% (w / v) sucrose;
(iv) about 0.01% to about 0.10% (w / v) of polysorbate 80; and (v) about 1 mM to about 20 mM L-methionine.
[7]
Formulation according to any one of claims 1 to 6, characterized in that it comprises about 8 mM to about 12 mM L-histidine buffer.
[8]
Formulation according to claim 6 or 7, characterized in that it comprises about 5 mM to about 10 mM L-methionine.
[9]
Formulation according to any one of claims 6 to 8, characterized in that it comprises polysorbate 80 at a weight ratio of about 0.02% w / v.
[10]
Formulation according to any one of claims 1 to 9, characterized in that it comprises about 10 mg / ml to about 100 mg / ml of the anti-TIGIT antibody or antigen-binding fragment thereof.
[11]
Formulation according to claim 10, characterized in that the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 10 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / ml, 75 mg / ml or 100 mg / ml.
[12]
12. Formulation according to any one of claims 1 to 11,
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3/5 characterized by the fact that it comprises about 25 mg / mL of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% polysorbate 80 and about 10 mM L-methionine.
[13]
Formulation according to any one of claims 1 to 11, characterized in that it comprises about 50 mg / ml of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose , about 0.02% polysorbate 80 and about 10 mM L-methionine.
[14]
Formulation according to any one of claims 1 to 11, characterized in that it comprises about 75 mg / ml of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose , about 0.02% polysorbate 80 and about 10 mM L-methionine.
[15]
Formulation according to any one of claims 1 to 11, characterized in that it comprises about 100 mg / ml of the anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose , about 0.02% polysorbate 80 and about 10 mM L-methionine.
[16]
Formulation according to any one of claims 1 to 15, characterized in that it has a pH of about 5.5 to 6.3.
[17]
17. Formulation according to claim 16, characterized by the fact that it has a pH of about 5.8 to 6.0.
[18]
Formulation according to any one of claims 1 to 17, characterized in that it additionally comprises an antiPD1 antibody or antigen-binding fragment thereof.
[19]
19. Formulation according to claim 18, characterized in that the anti-human PD-1 antibody or antigen-binding fragment thereof comprises three light chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and three heavy chain CDRs of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
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4/5
[20]
Formulation according to claim 18 or 19, characterized in that the anti-human PD-1 antibody or antigen-binding fragment thereof comprises a light variable region comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy variable region comprising the amino acid sequence shown in SEQ ID NO: 9.
[21]
21. Formulation according to any of claims 16 to 19, characterized in that it comprises an anti-human PD-1 antibody that is pembrolizumab.
[22]
22. Formulation according to any one of claims 18 to 21, characterized in that the ratio of the anti-PD1 antibody to the anti-TIGIT antibody is 1: 1.
[23]
23. Formulation according to any one of claims 18 to 22, characterized in that it comprises about 20 mg / ml of the anti-PD1 antibody, about 20 mg / ml of the anti-TIGIT antibody, 10 mM Lhistidine buffer , about 7% w / v sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine.
[24]
24. Formulation according to any one of claims 1 to 23, characterized in that it additionally comprises a chelator.
[25]
25. Formulation according to claim 24, characterized by the fact that the chelator is DTPA.
[26]
26. Formulation according to any one of claims 1 to 25, characterized in that it is contained in a glass vial or an injection device.
[27]
27. Formulation according to any one of claims 1 to 26, characterized in that it is a liquid formulation, which is frozen at least below -70 ° C, or is a solution reconstituted from a lyophilized formulation.
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5/5
[28]
28. Formulation according to any of claims 1 to 27, characterized by the fact that after 12 months at 5 ° C:
(i) the% monomer of the anti-TIGIT antibody is> 95% as determined by size exclusion chromatography;
(ii) the% of heavy and light chain of the anti-TIGIT antibody is> 90% as measured by reducing CE-SDS;
(iii) the% of heavy and light chain of the anti-TIGIT antibody is> 95%, as measured by reducing CE-SDS;
(iv) the% of intact IgG of the anti-TIGIT antibody is> 90% as measured by non-reducing CESDS; and / or (v) the% intact IgG of the anti-TIGIT antibody is> 95% as measured by non-reducing CESDS.
[29]
29. Use of the formulation defined in any of claims 1 to 28, characterized by the fact that it is for preparing a drug for the treatment of cancer or for the treatment of chronic infection.
[30]
30. Invention of a product, process, system, kit or use, characterized by the fact that it comprises one or more elements described in the present patent application.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762500278P| true| 2017-05-02|2017-05-02|

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PCT/US2018/030516|WO2018204405A1|2017-05-02|2018-05-01|Stable formulations of anti-tigit antibodies alone and in combination with programmed death receptor 1antibodies and methods of use thereof|

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