bonding agents

专利摘要:
The invention relates to antibody molecules and their antigen-binding fractions that specifically bind to CD47 (Differentiation Cluster 47, also known as an integrin-associated protein [IAP]). In aspects of the invention, the anti-CD47 antibody molecules and their antigen-binding fractions specifically bind to human CD47 and CD47 of the cynomolgus monkey. Medical uses of the anti-CD47 antibody molecules and antigen-binding fractions of the invention are disclosed. The anti-CD47 antibody molecules and the antigen-binding fractions of the invention represent modified and optimized binding molecules compared to a murine / humanized anti-CD47 anti-CD47 antibody described in WO2014 / 093678A2.
公开号:BR112020003306A2
申请号:R112020003306-5
申请日:2018-08-17
公开日:2020-08-25
发明作者:William James Jonathan Finlay
申请人:Ultrahuman Four Limited；
IPC主号:

专利说明:

[001] [001] The invention relates to antibody molecules that specifically bind to CD47 (Differentiation Cluster 47, also known as integrin-associated protein [IAP]) and their medical uses. BACKGROUND OF THE INVENTION
[002] [002] CD47 (also known as integrin-associated protein [IAP]) is a transmembrane protein that belongs to the immunoglobulin superfamily and binds to several known partners, including: membrane integrins, thrombospondin-1 (TSP-1) and protein alpha signal regulator (SIRPα) CD47 is associated with a variety of cellular processes, including apoptosis, cell proliferation, adhesion and migration, and mainly plays a key role in immune and angiogenic responses. CD47-SIRPα signaling is a critical molecular interaction that inhibits the activation of phagocytosis by macrophages and other myeloid cells. This promotes the survival of tumor cells and therefore acts as an immune checkpoint specific to the myeloid lineage.
[003] [003] Preclinical evidence suggests that blocking CD47-SIRPα signaling may increase the phagocytic activity of macrophages and inhibit the growth of xenografts in several experimental models of hematological and solid malignancies. As the activity of macrophages is also a recognized factor in the biology of tissue remodeling associated with inflammation, such as tissue fibrosis and the formation of atherosclerotic plaques, the CD47-SIRPα signaling axis also has considerable therapeutic potential in non-cancerous diseases. Therefore, anti-CD47 mAbs have the potential to act as immunotherapeutic agents in cancer and other contexts, in addition to amplifying the effectiveness of currently established therapies.
[004] [004] Most of the currently approved antibody therapies are derived from immunized rodents.
[005] [005] Although humanized antibodies via CDR grafting and reverse mutations have been shown to induce lower rates of immune response in the clinic compared to those with fully murine v domains, humanized antibodies using this basic graft method still carry significant risks of clinical development due to instability of physical potential and the reasons for immunogenicity still housed in the grafted CDR loops.
[006] [006] The ideal humanized antagonist anti-CD47 antibody would therefore have as many identical residues as possible in the v domains as found in well-characterized human germline sequence structures and CDRs. Townsend et al. (2015; PNAS 112: 15354-15359) describe a method for generating antibodies in which
[007] [007] The germline alignment of CDR is, therefore, a complex multifactorial problem, since several functional properties of the molecule should preferably be maintained, including in this case: specificity of target binding, affinity to CD47 of test species in humans and animals ( for example, cynomolgus monkey, also known as the crab-eating monkey, ie Macaca fascicularis), the biophysical stability of the ve domain, or the expression of IgG.
[008] [008] WO2014 / 093678A2 describes an antagonist murine anti-CD47 IgG molecule called "VxP037" and also the preparation of humanized forms of VxP037. These humanized forms of VxP037 were produced using classical humanization techniques, that is, grafting murine CDRs defined by Kabat into human heavy and light chain structure sequences, with some of the human structure residues being potentially reversed mutated to the VxP037 murine residues correspondingly positioned. For the reasons mentioned above, these humanized forms of VxP037 described in WO2014 / 093678A2 are not ideal.
[009] [009] The present invention provides a series of optimized anti-CD47 antibodies and their medical uses. SUMMARY OF THE INVENTION
[010] [010] In accordance with one aspect of the invention, an antibody molecule is provided which specifically binds to human CD47 and, optionally, also to cynomolgus monkey CD47 and / or mouse CD47, or a fraction thereof. antigen, wherein the antibody molecule or antigen binding fraction comprises a heavy chain variable region with: an HCDR1 with amino acids in sequence in the following order: GYT or any amino acid (for example, S, N or R) -FT or a conservative substitution of TN or a conservative substitution of NYYI or a conservative substitution of IF or any amino acid (for example, V or G) (SEQ ID NO: 1); an HCDR2 with amino acids in sequence in the following order: M or a conservative MGI substitution or any amino acid (for example, N, V or D) -IN or any amino acid (for example, Y) -PV or any acidic amino acid (for example , G or F) -D or a conservative substitution of DG or a conservative substitution of GDTN or a conservative substitution of N (for example, R) -Y or a conservative substitution of YN or a conservative substitution of N (for example, S ) -PSFQG (SEQ ID NO: 2); and an HCDR3 with amino acids in sequence in the following order: GGY or any amino acid (for example, H, I, Q or F) -T or any amino acid (for example, V or I) -M or any amino acid (for example, T , R, P, A or L) -D or any amino acid (for example, G) -R or any amino acid (for example, Q, N, Y, S, W, K, A, E, F, H, I , L, M, T or V) (SEQ ID NO: 3).
[011] [011] In aspects of the invention, the HCDR1 of the antibody molecule or antigen binding fraction may exclude the GYTFTNYYVF sequence (SEQ ID NO: 4) (murine / humanized HCDR1 VxP037 antibody disclosed in WO2014 / 093678A2) and / or HCDR3 from the antibody to the antigen binding molecule or fraction can exclude the sequence
[012] [012] The antibody molecule or antigen-binding fraction can further comprise a variable region of the light chain with: an LCDR1 with amino acids in sequence in the following order: RSSQ or a conservative substitution of QSL or a conservative substitution of LL or a substitution conservative LHSN or any amino acid (for example, Q, S, T, A or G) or a conservative N substitution (for example, Q, S, T or G) -G or a conservative G substitution (for example, A) -Y or any amino acid (for example, N or S) -T or a conservative substitution for T (for example, N) -YLH or any amino acid (for example, D) (SEQ ID NO: 6); an LCDR2 with amino acids in sequence in the following order: K or any amino acid (for example, L or M) -V or any amino acid (for example, G) -SN or any amino acid (for example, Y) -RL or any amino acid ( for example, F, A or S) -S (SEQ ID NO: 7); and an LCDR3 with amino acids in sequence in the following order: F or any amino acid (for example, L, M, S, T or V) -QQ or any amino acid (for example, N, A, T or S) -T or any amino acid (for example, L, M or I) -H or a conservative substitution for HT or any amino acid (for example, V, I, A or F) -P or any amino acid (for example, L) -R or any amino acid (e.g., W) -T (SEQ ID NO: 8).
[013] [013] In aspects of the invention, the LCDR1 of the antibody molecule or antigen-binding fraction can exclude the sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9) (murine antibody VxP037 / humanized LCDR1 disclosed in WO2014 / 093678A2) and / or LCDR2 do antibody molecule or antigen binding fraction may exclude the KVSYRFS sequence (SEQ ID NO: 10) (murine antibody VxP037 / humanized LCDR2 disclosed in WO2014 / 093678A2) and / or the LCDR3 of the antibody molecule or antigen binding fraction exclude the sequence SQNTHVPRT (SEQ ID NO: 11) (murine antibody VxP037 / humanized LCDR3 disclosed in WO2014 / 093678A2).
[014] [014] The CDR sequences above are defined using the "Unified" definition, as set out in Table 1 and described below. Alternatively, the CDR sequences in the present invention can be defined using the shortest definition of "AHo" (see Table 1), which is based on structural biology and aims to unify the nomenclature for all immunoglobulin v domains.
[015] [015] Using the shorter definition of CDR "AHo", the invention in one respect provides an antibody molecule that specifically binds to human CD47 and, optionally, also to CD-47 monkey and / or mouse CD47, or to a fraction of the same antigen-binding, where the antibody molecule or antigen-binding fraction comprises a variable region of the heavy chain with: an HCDR1 with amino acids in sequence in the following order: GSGYT or any amino acid (for example, S , N or R) - FT or a conservative substitution of TN or a conservative substitution of NYY (SEQ ID NO: 12); an HCDR2 with amino acids in sequence in the following order: IN or any amino acid (for example, Y) -PV or any amino acid (for example, G or F) -D or a conservative DG substitution or a conservative GDTN substitution or a substitution conservative N (for example, R) -Y or a conservative substitution of YN or a conservative substitution of N (for example, S) -PSFQG (SEQ ID NO: 13); and an HCDR3 with amino acids in sequence in the following order: GGY or any amino acid (for example, H, I, Q or F) -T or any amino acid (for example, V or I) -M or any amino acid (for example, T , R, P, A or L) -D or any amino acid (for example, G) (SEQ ID NO: 14).
[016] [016] Using the definition of AHo, the HCDR1 of the antibody molecule or antigen binding fraction can exclude the sequence GSGYTFTNYY (SEQ ID NO: 15) (murine antibody VxP037 / humanized HCDR1 disclosed in WO2014 / 093678A2) and / or the HCDR3 of the antibody molecule or the antigen binding fraction can exclude the GGYTMD sequence (SEQ ID NO: 16) (murine VxP037 / humanized HCDR3 antibody disclosed in WO2014 / 093678A2).
[017] [017] The antibody molecule or antigen-binding fraction may further comprise a variable region of the light chain with CDRs defined using the definition of AHo as follows: an LCDR1 with amino acids in sequence in the following order: SSQ or a conservative substitution of QSL or a conservative LL substitution or a conservative LHSN substitution or any amino acid (for example, Q, S, T, A or G) or a conservative N substitution (for example, Q, S, T or G) -G or a conservative substitution of G (for example, A) -Y or any amino acid (for example, N or S) -T or a conservative substitution of T (for example, N) -Y (SEQ ID NO: 17); an LCDR2 with amino acids in sequence in the following order: K or any amino acid (for example, L or M) -V or any amino acid (for example, G) -SN or any amino acid (for example, Y) -RL or any amino acid ( for example, F, A or S) -S (SEQ ID NO: 7); and an LCDR3 with amino acids in sequence in the following order: Q or any amino acid (for example, N, A, T or S) -T or any amino acid (for example, L, M or I) -H or a conservative HT replacement or any amino acid (for example, V, I, A or F) -P or any amino acid (for example, L) -R or any amino acid (for example, W) (SEQ ID NO: 18).
[018] [018] Using the definition of AHo, in aspects of the invention, the LCDR1 of the antibody molecule or antigen binding fraction can exclude the sequence SSQSLVHSNGNTY (SEQ ID NO: 19) (murine antibody VxP037 / humanized LCDR1 disclosed in WO2014 / 093678A2 ) and / or the LCDR2 of the antibody molecule or antigen binding fraction may exclude the KVSYRFS sequence (SEQ ID NO: 10) (murine antibody VxP037 / humanized LCDR2 disclosed in WO2014 / 093678A2) and / or the LCDR3 of the antibody molecule or antigen binding fraction may exclude the NTHVPR sequence (SEQ ID NO: 20) (murine VxP037 / humanized LCDR3 antibody disclosed in WO2014 / 093678A2).
[019] [019] An immunoconjugate comprising the antibody molecule or its antigen-binding fraction, as defined herein, attached to a therapeutic agent is also provided according to the invention.
[020] [020] In another aspect, the invention provides a nucleic acid molecule that encodes the antibody molecule or its antigen-binding fraction, as defined herein.
[021] [021] A vector further comprising the nucleic acid molecule of the invention is provided.
[022] [022] A host cell comprising the nucleic acid molecule or vector of the invention as defined herein is also provided.
[023] [023] In a further aspect, a method is provided to produce an anti-CD47 antibody and / or an antigen binding fraction, comprising culturing the host cell of the invention under conditions that result in the expression and / or production of the antibody and / or its antigen-binding fraction and antibody isolation and / or its antigen-binding fraction from the host cell or culture.
[024] [024] In another aspect of the invention, a pharmaceutical composition is provided comprising the antibody molecule or its antigen-binding fraction of the invention, as defined herein, or the immunoconjugate of the invention, as defined herein, or the nucleic acid molecule of the invention. invention as defined herein, or the vector of the invention as defined herein.
[025] [025] In addition, a method is provided to improve an immune response in an individual, comprising administering an effective amount of the antibody molecule or its binding fraction to the antigen of the invention, as defined herein, or the immunoconjugate of the invention, as defined herein, either the nucleic acid molecule of the invention as defined herein, or the vector of the invention as defined herein, or the pharmaceutical composition of the invention as defined herein.
[026] [026] In a further aspect, a method is provided to treat or prevent cancer in an individual, comprising administering an effective amount of the antibody molecule or its antigen-binding fraction as defined herein, or the immunoconjugate of the invention, as defined herein, either the nucleic acid molecule of the invention as defined herein, or the vector of the invention as defined herein, or the pharmaceutical composition of the invention as defined herein.
[027] [027] The invention also provides an antibody molecule or its binding fraction to the antigen of the invention, as defined herein, or the immunoconjugate of the invention, as defined herein, or the nucleic acid molecule of the invention, as defined herein, or the vector of the invention, as defined herein, or the pharmaceutical composition of the invention, as defined herein, for use in the treatment of cancer.
[028] [028] In another aspect, the invention provides the antibody molecule, or its antigen-binding fraction, or the immunoconjugate, or the nucleic acid molecule, or the vector for use, or the method of treatment of the invention, as defined here, for separations, sequential or sequential.
[029] [029] In an additional aspect, use is made of an antibody molecule or an antigen-binding fraction of the invention, as defined herein, or an immunoconjugate of the invention, as defined herein, or a nucleic acid molecule of the invention, as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the manufacture of a medicament for the treatment of cancer.
[030] [030] The invention also provides a method for the treatment or prevention of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease in an individual, comprising administering an effective amount of the antibody molecule or its fraction of binding to the antigen, as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein.
[031] [031] Autoimmune disease or inflammatory disease can be selected in all aspects of the group consisting of: arthritis, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave's disease and Hashimoto's thyroiditis and ankylosing spondylitis.
[032] [032] Ischemia-reperfusion injury in all aspects can occur in organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resections and reconstructive surgery, replacement from an appendix or other body part, skin graft or trauma.
[033] [033] Also provided is an antibody molecule or its antigen-binding fraction, as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein, for use in the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
[034] [034] In addition, the use of an antibody molecule or its antigen-binding fraction, as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, is provided , or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
[035] [035] The invention also provides a method for the treatment or prevention of cardiovascular disease or fibrotic disease in an individual, comprising administering an effective amount of the antibody molecule or its antigen-binding fraction, as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein.
[036] [036] An antibody molecule or its antigen-binding fraction as defined herein is also provided, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein, for use in the treatment of cardiovascular disease or fibrotic disease.
[037] [037] In addition, use is made of an antibody molecule or its antigen-binding fraction, as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein , or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of an ischemia-reperfusion injury, an autoimmune disease, an inflammatory disease or a fibrotic disease.
[038] [038] Cardiovascular disease in any aspect of the invention can be for example coronary heart disease or atherosclerosis.
[039] [039] Fibrotic disease in any aspect of the invention can be selected from the group consisting of myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis and asthma.
[040] [040] The invention also provides a method for producing an antibody molecule that specifically binds to
[041] [041] The method may comprise an additional step of producing additional clones based on the clones selected in step (4), for example, based on additional exploratory mutagenesis at specific positions in the CDRs of the clones selected in step (4), for improve humanization and / or minimize the content of the human T cell epitope and / or improve the manufacturing properties in the antibody molecule or its antigen-binding fraction produced in step (5).
[042] [042] The method may comprise an additional step of assessing the immunogenicity of one or more domains v in the clones selected in step (4) or in the antibody molecule produced in step (5) and, optionally, generating one or more additional mutations, for example, in a CDR and region of the structure, to reduce immunogenicity. Immunogenicity can be assessed by identifying the location of T cell epitopes, for example, using in silico technologies as described herein. BRIEF DESCRIPTION OF THE FIGURES
[043] [043] Figure 1. Direct binding ELISA of library-derived anti-CD47 scFvs against human and mouse CD47-Fc proteins. The clones were derived from 3 separate phage selection branches (A shows branch A periprep ELISA; B shows branch B periprep ELISA; and C shows branch C periprep ELISA), where phage populations were selected on CD47- Biotinylated human Fc, mouse and / or cynomolgus monkey in each round. After each round of selection, clones derived from the library (black circles) were screened against human and mouse CD47-Fc. The mean values ± SD in each round are represented in gray bars. In each graph, the X axis shows the selection circle ("R"), with "H" denoting human mouse and "M" and the Y axis showing a connection signal (OD 450nm).
[044] [044] Figure 2. Tolerance analysis of CDR residues for mutation in the germline. A graph of murine amino acid retention frequencies in the CDRs of the ELISA positive population of 854 single scFv clones is shown for the VH (A) and VL (B) domains, respectively.
[045] [045] Figure 3. Direct titration ELISA for binding IgG to human, mouse and cyan CD47-Fc proteins.
[046] [046] Figure 4. CD47-Fc-SIRPα competition assay based on ELISA. The ELISA binding signal for human CD47-Fc proteins (A), chino (B) and mouse (C) to plate-bound human SIRPα was examined in the presence of major derivatives from the titled competitor library: A-D5, G -B6, D-H3 and VH-A1 / VL-B1 in IgG1null format, Isotype IgG1 as negative control, plus mVH / mVL in IgG1null format as positive control. All library-derived IgGs and mVH / mVL demonstrated a concentration-dependent reduction in the binding of human, murine and cyano CD47-Fc proteins, suggesting the maintenance of a shared epitope. Notably, clone A-D5 exhibited significantly increased potency in neutralizing mouse CD47 compared to mVH / mVL and VH-A1 / VL-B1 did not show the ability to neutralize murine CD47 activity. Neither the designer clone MH nor the TTP exhibited any sign of neutralization and are not plotted here, for clarity. In each graph, the X axis shows the concentration of antibodies in nM and the Y axis shows the binding signal (OD 450 nm). In the figure captions, "IC" refers to isotype control.
[047] [047] Figure 5. Analysis of binding specificity for priority clones ("lead clones") prioritized. Risk of off-target homologous binding for mVH / mVL in the IgG1 (A) and IgG1null (B) format and principal derivatives of the A-D5 (C), VH-A1 / VL-B1 (D), F-E7 library ( E), D-H3 (F) and G-B6 (G) in IgG1 format were examined by direct ELISA in CD47-Fc orthologists and a panel of 14 proteins from the human immunoglobulin superfamily labeled on each X axis ("B" if refers to blank). Binding to human, cino and murine CD47-Fcs (h / c / mCD47-Fc) was performed at an IgG concentration of 1 µg / ml. Binding to all other proteins was performed at an IgG concentration of 10 µg / ml. In each graph, the Y axis shows a connection signal (OD 450 nm). For almost all IgGs, binding was observed only to hCD47-Fc,
[048] [048] Figure 6. Cytometric flow connection to human and CD47 + CHO-K1 cells. The commercial anti-CD47 antibody MS1991, human IgG1 ("I IgG1") and IgG4 ("I IgG4") isotype controls, major library derived IgGs in the IgG1null ("IgG1N") and IgG4 (S228P) formats were examined for specific binding in cyto-transfected CHO-K1 cells (A), human-transfected CHO-K1 cells (B) and wild-type CHO-K1 cells (by weight, i.e., not transfected) (C). IgGs were tested in concentrations ranging from 24-100,000 ng / ml. Concentration-dependent binding was observed against human cell lines and cyano for all antibodies specific for CD47, but not for Isotype controls. Low-level binding signals above the background were observed against wild-type CHO-K1 cells for most antibodies, with significantly stronger signal for mVH / mVL-derived IgGs and particularly low signal for VH-A1 / VL- IgGs B1.
[049] [049] Figure 7. Cytometric flow test for binding to human HL60 cells. Commercial anti-CD47 antibody MS1991, isotype controls IgG1 and human IgG4 ("I IgG1" and "I IgG4", respectively), main derivatives of the library in both IgG1null ("IgG1N) and IgG4 (S228P) formats were examined for specific binding in HL60 cells. IgGs were tested in concentrations ranging from 24-100000 ng / ml.
[050] [050] Figure 8. ELISAs at risk of development. This assay showed that antibodies A-D5, G-B6, D-H3, VH-A1 / VL-B1 and mVH / mVL in IgG1null form exhibit little or no binding to the negatively charged biomolecules Insulin (A), double-stranded DNA (dsDNA) (B) and single-stranded DNA (ssDNA) (C). In each graph, the X axis shows the IgG concentration in µg / ml and the Y axis shows the binding signal (OD 450 nm).
[051] [051] Figure 9. Direct titration ELISA for the binding of designer IgGs to human, mouse and cyan CD47-Fc proteins. Chimeric anti-CD47 clones (mVH / mVL), designer clones derived from A-D5 in human IgG1null format, were titrated (in µg / ml) in a direct binding ELISA against human CD47-Fc proteins (A), cino ( B) and mouse (C).
[052] [052] Figure 10. CD47-Fc-SIRPα competition test based on ELISA for designer IgGs. The ELISA binding signal for human CD47-Fc proteins (A), cyino (B) and mouse (C) to plate-bound human SIRPα was examined in the presence of competing designer IgGs titrated in the IgG1null format, plus the IgG1 isotype as a negative control (represented by “IgG1NI”) and mVH / mVL in the IgG1null format as a positive control. In each graph, the X axis shows the IgG concentration in nM and the Y axis shows the binding signal (OD 450 nm). Notably, several clones derived from A-D5 again exhibited significantly increased potency in neutralizing mouse CD47 compared to mVH / mVL. Neither the designer clone A-D5.7 nor A-D5.10 exhibited any sign of neutralization in the orthologists for whom they showed weak ELISA-binding signal and are not plotted here, for clarity.
[053] [053] Figure 11. Direct titration ELISA for designer IgGs derived from A-D5.4 that bind to human, mouse and cino CD47-Fc proteins. Chimeric anti-CD47 clones (mVH / mVL), clones derived from designer A-D5.4, in human IgG1null format, were titrated (in µg / ml) in a direct binding ELISA against human CD47-Fc proteins (A) , cino (B) and mouse (C). In each graph, the X axis shows the IgG concentration in µg / ml and the Y axis shows the binding signal (OD 450 nm). All clones showed binding activity against all 3 CD47 orthologists.
[054] [054] Figure 12. CD47-Fc-SIRPα competition assay based on ELISA for designer IgGs. The ELISA binding signal for human CD47-Fc (A), cyino (B) and mouse (C) proteins to plate-bound human SIRPα was examined in the presence of competing designer IgGs titrated in the IgG1null format, plus the IgG1 isotype as negative control (represented by “IgG1NI”) and mVH / mVL in IgG1null format as a positive control. In each graph, the X axis shows the IgG concentration in nM and the Y axis shows the binding signal (OD 450 nm). Notably, several clones derived from A-D5.4 again exhibited significantly increased potency in neutralizing mouse CD47 compared to mVH / mVL.
[055] [055] Figure 13. Binding specificity analyzes for designer clones A-D5.4 and A-D5.16. The risk of off-target homologous binding for A-D5.4 (A) and A-D5.16 (B) in IgG1null format was examined by direct ELISA in CD47-Fc orthologists and a panel of 14 proteins from the immunoglobulin superfamily (as indicated on each X axis; "B" refers to white). Binding to all proteins was performed at an IgG concentration of 10 µg / ml. In each graph, the Y axis shows a connection signal (OD 450 nm). For both IgGs, binding was observed only to hCD47-Fc, mCD37-Fc and cCD47-Fc. No over-the-bottom binding was observed for any other human protein.
[056] [056] Figure 14. Development risk ELISAs for designer clones A-D5.4 and A-D5.16. This assay showed that antibodies A-D5.4 and A-D5.16 in the form of IgG1null exhibit low binding (below the negative control, Ustekinumab) to the negatively charged biomolecules Insulin (A), double-stranded DNA (dsDNA) (B ) and single stranded DNA (ssDNA) (C). In each graph, the X axis shows the IgG concentration in µg / ml and the Y axis shows the binding signal (OD 450 nm). The strong off-target binding to these molecules, as observed for the Bococizumab and Briakinumab analogs, has been shown to be a high risk indicator of low clinical performance of therapeutic antibodies.
[057] [057] Figure 15. Flow cytometric binding of designer IgGs derived from the library to CHO-K1 cells. Commercial MS1991 anti-CD47 antibody, IgG1 and human IgG4 isotype controls (represented by "I IgG1" and "I IgG4", respectively) and main IgG A-D5, A-D5.4 and A-D5.16, both in IgG1null and IgG4 formats were examined for specific binding in wild-type (ie, non-transfected) CHO-K1 cells. IgGs were tested in concentrations ranging from 24 to 25,000 ng / ml. Concentration-dependent binding was observed for the parental antibody mVH / mVL in the IgG1null and IgG4 formats, but only weak or no binding was observed in the Isotype, MS1991 and IgGs controls A-D5, A-D5.4 and A-D5.16 in both IgG formats. In each graph, the X axis shows the IgG concentration in ng / ml, and the Y axis shows MFI.
[058] [058] Figure 16. Cytometric flow test for binding to human HL60 cells. Commercial anti-CD47 antibody MS1991, human IgG1 and IgG4 isotype controls (represented by "I IgG1" and "I IgG4", respectively), major IgGs in IgG1null and IgG4 (S228P) formats were examined for specific binding to HL60 cells. IgGs were tested in concentrations ranging from 24-100000 ng / ml.
[059] [059] Figure 17. T cell epitope peptide content in the v domains of the main antibody. The v domains of the mVH / mVL antibodies, A-D5, A-D5.4 A-D5.16 and A-D5.16-DI were examined for the presence of germline (GE), high external affinity (HAF), low external affinity (LAF) and TCED + T cell receptor epitopes. The VH and VL domains of mVH / mVL have been found to contain multiple high-risk human T cell epitopes and few germline epitopes. In all major clones, the content of the high-risk epitope was significantly reduced and the content of the germline epitope improved significantly.
[060] [060] Figure 18. Direct titration ELISA for designer IgGs A-D5.16 and A-D5.16-DI that bind to human, mouse and cyano CD47-Fc proteins. The chimeric anti-CD47 clones (mVH / mVL), designer A-D5.16 and A-D5.16-DI in the human IgG1null format were titrated (in µg / ml) in a direct binding ELISA against CD47-Fc proteins of human (A), cino (B) and mouse (C). All clones showed binding activity against all 3 CD47 orthologists. In each graph, the X axis shows the IgG concentration in µg / ml, and the Y axis shows the binding signal (OD 450 nm).
[061] [061] Figure 19. CD47-Fc-SIRPα S competition test based on ELISA for designer IgGs. The ELISA binding signal for human CD47-Fc (A), cyino (B) and mouse (C) proteins to plate-bound human SIRPα was examined in the presence of competing titrated IgGs in the IgG1 format, plus the isotype IgG1 as a negative control and mVH / mVL in the IgG1null format as a positive control. In each graph, the X axis shows the concentration of antibodies in nM and the Y axis shows the binding signal (OD 450 nm).
[062] [062] Figure 20. Phagocytosis analyzes by flow cytometry. (A) Flow cytometric analysis of phagocytosis of CSF-labeled HL60 cells by human CD14 + macrophages was performed in multiple concentrations (as shown on the X axis) for clones A-D5, A-D5.4. A-D5.16 and mVH / mVL in IgG4 format (S228P) and additionally A-D5 in IgG1null format (represented by "IgG1 A-D5N"). The X axis shows the concentration of antibodies (µg / ml) and the Y axis shows% of cells that are CFSE + and CD14 +. (B) The analysis was then repeated on several human macrophage donors for A-D5 and mVH / mVL in IgG4 format, at a standard concentration of 10 µg / ml. The X axis shows the donor number and the Y axis shows% of cells that are CFSE + and CD14 +. "V" indicates vehicle. DETAILED DESCRIPTION OF THE INVENTION
[063] [063] In accordance with a first aspect of the invention, an antibody molecule is provided which specifically binds to human CD47 and, optionally, also to cynomolgus monkey CD47 and / or mouse CD47, or a fraction of antigen binding to even, where the antibody molecule or antigen-binding fraction comprises a variable region of heavy chain with: an HCDR1 with amino acids in sequence in the following order: GYT or any amino acid (for example, S, N or R) -FT or a conservative substitution of TN or a conservative substitution of NYYI or a conservative substitution of IF or any amino acid (for example, V or G) (SEQ ID NO: 1); an HCDR2 with amino acids in sequence in the following order: M or a conservative MGI substitution or any amino acid (for example, N, V or D) -IN or any amino acid (for example, Y) -PV or any acidic amino acid (for example , G or F) -D or a conservative substitution of DG or a conservative substitution of GDTN or a conservative substitution of N (for example, R) -Y or a conservative substitution of YN or a conservative substitution of N (for example, S ) -PSFQG (SEQ ID NO: 2); and an HCDR3 with amino acids in sequence in the following order: GGY or any amino acid (for example, H, I, Q or F) -T or any amino acid (for example, V or I) -M or any amino acid (for example, T , R, P, A or L) -D or any amino acid (for example, G) -R or any amino acid (for example, Q, N, Y, S, W, K, A, E, F, H, I , L, M, T or V) (SEQ ID NO: 3).
[064] [064] In aspects of the invention, the HCDR1 of the antibody molecule or antigen-binding fraction may exclude the sequence GYTFTNYYVF (SEQ ID NO: 4) (murine antibody VxP037 / humanized HCDR1 disclosed in WO2014 / 093678A2) and / or HCDR3 from the antibody to the antigen binding molecule or fraction can exclude the GGYTMDY sequence (SEQ ID NO: 5) (murine VxP037 / humanized HCDR3 antibody disclosed in WO2014 / 093678A2).
[065] [065] The antibody molecule or its antigen-binding fraction according to the invention can further comprise a variable region of the light chain with: an LCDR1 with amino acids in sequence in the following order: RSSQ or a conservative substitution of QSL or a substitution conservative LL or a conservative LHSN substitution or any amino acid (for example, Q, S, T, A or G) or a conservative N substitution (for example, Q, S, T or G) -G or a conservative substitution of G (for example, A) -Y or any amino acid (for example, N or S) -T or a conservative T substitution (for example,
[066] [066] In aspects of the invention, the LCDR1 of the antibody molecule or antigen binding fraction can exclude the sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9) (murine antibody VxP037 / humanized LCDR1 disclosed in WO2014 / 093678A2) and / or LCDR2 do antibody molecule or antigen binding fraction may exclude the KVSYRFS sequence (SEQ ID NO: 10) (murine antibody VxP037 / humanized LCDR2 disclosed in WO2014 / 093678A2) and / or the LCDR3 of the antibody molecule or antigen binding fraction delete string
[067] [067] The CDR sequences above are defined using the "Unified" definition, as set out in Table 1.
[068] [068] Using the shorter definition of "AHo", the invention, in one aspect, provides an antibody molecule that specifically binds to human CD47 and, optionally, also to cynomolgus monkey CD47 and / or mouse CD47, or an antigen-binding fraction, where the antibody molecule or antigen-binding fraction comprises a variable region of the heavy chain with: an HCDR1 with amino acids in sequence in the following order: GSGYT or any amino acid (for example, S, N or R) - FT or a conservative substitution of TN or a conservative substitution of NYY (SEQ ID NO: 12); an HCDR2 with amino acids in sequence in the following order: IN or any amino acid (for example, Y) -PV or any amino acid (for example, G or F) -D or a conservative DG substitution or a conservative GDTN substitution or a substitution conservative N (for example, R) -Y or a conservative substitution of YN or a conservative substitution of N (for example, S) -PSFQG (SEQ ID NO: 13); and an HCDR3 with amino acids in sequence in the following order: GGY or any amino acid (for example, H, I, Q or F) -T or any amino acid (for example, V or I) -M or any amino acid (for example, T , R, P, A or L) -D or any amino acid (for example, G) (SEQ ID NO: 14).
[069] [069] Using the definition of AHo, the HCDR1 of the antibody molecule or antigen binding fraction can exclude the sequence GSGYTFTNYY (SEQ ID NO: 15) (murine antibody VxP037 / humanized HCDR1 disclosed in WO2014 / 093678A2) and / or the HCDR3 of the antibody molecule or the antigen binding fraction can exclude the GGYTMD sequence (SEQ ID NO: 16) (murine VxP037 / humanized HCDR3 antibody disclosed in WO2014 / 093678A2).
[070] [070] The antibody molecule or antigen-binding fraction can further comprise a variable region of the light chain with: an LCDR1 with amino acids in sequence in the following order: SSQ or a conservative substitution of QSL or a conservative substitution of LL or a substitution conservative LHSN or any amino acid (for example, Q, S, T, A or G) or a conservative N substitution (for example, Q, S, T or G) -G or a conservative G substitution (for example, A) -Y or any amino acid (for example, N or S) -T or a conservative substitution for T (for example, N) -Y (SEQ ID NO: 17); an LCDR2 with amino acids in sequence in the following order: K or any amino acid (for example, L or M) -V or any amino acid (for example, G) -SN or any amino acid (for example, Y) -RL or any amino acid ( for example, F, A or S) -S (SEQ ID NO: 7); and an LCDR3 with amino acids in sequence in the following order: Q or any amino acid (for example, N, A, T or S) -T or any amino acid (for example, L, M or I) -H or a conservative HT replacement or any amino acid (for example, V, I, A or F) -P or any amino acid (for example, L) -R or any amino acid (for example, W) (SEQ ID NO: 18).
[071] [071] Using the definition of AHo, in aspects of the invention, the LCDR1 of the antibody molecule or antigen binding fraction can exclude the sequence SSQSLVHSNGNTY (SEQ ID NO: 19) (murine antibody VxP037 / humanized LCDR1 disclosed in WO2014 / 093678A2 ) and / or the LCDR2 of the antibody molecule or antigen binding fraction may exclude the KVSYRFS sequence (SEQ ID NO: 10) (murine antibody VxP037 / humanized LCDR2 disclosed in WO2014 / 093678A2) and / or the LCDR3 of the antibody molecule or antigen binding fraction may exclude the NTHVPR sequence (SEQ ID NO: 20) (murine VxP037 / humanized LCDR3 antibody disclosed in WO2014 / 093678A2).
[072] [072] As elaborated here, the present inventors were able for the first time to generate a number of optimized anti-CD47 antibody molecules using CDR sequences derived from the murine anti-CD47 antibody VxP037 disclosed in WO2014 / 093678A2. In the embodiments of the present invention, these antibody molecules have been selected to have binding specificity to both human CD47 and cynomolgus monkey CD47 and, for some clones, also to mouse CD47 (to facilitate studies in animal test species). The further refinement of the optimized antibody molecules, as described here, provided improved binding to the mouse CD47 orthologist, improved potency in neutralizing mouse CD47-SIRPα signaling, improved variable domain stability, high expression yields and / or reduced immunogenicity. For example, we demonstrate here that the murine anti-CD47 antibody VxP037 progenitor molecule carries two main immunogenicity risks in LCDR1 and LCDR2 that will be performed with classic humanization techniques (as used in WO2014 / 093678A2), but which are optimization of antibody molecules as described herein.
[073] [073] The antibody molecule or antigen-binding fraction of the present invention may have improved in silico immunogenicity compared to an antibody molecule comprising the CDR sequences of SEQ ID NOs: 4 (HCDR1), 123 (HCDR2), 5 (HCDR3), 9 (LCDR1), 10 (LCDR2) and 11 (LCDR3).
[074] [074] The antibody molecule or antigen-binding fraction of the present invention may lack binding to the hamster's CD47 or exhibit reduced binding to the hamster's CD47 compared to an antibody molecule comprising the CDR sequences of SEQ ID NOs: 4 (HCDR1), 123 (HCDR2), 5 (HCDR3), 9 (LCDR1), 10 (LCDR2) and 11 (LCDR3). For example, the antibody molecule or antigen-binding fraction of the present invention may exhibit no binding to CHO cells as measured by flow cytometry, or reduced binding to CHO cells as measured by flow cytometry, compared to an antibody comprising the CDR sequences of SEQ ID NOs: 4 (HCDR1), 123 (HCDR2), 5 (HCDR3), 9 (LCDR1), 10 (LCDR2) and 11 (LCDR3). As shown in Fig. 15, the antibody molecules representative of the present invention have little or no cross-reactivity with CHO cells, while the original murine v domains of the mVH / mVL clone in the IgG1null or IgG4 format conduct a strong, concentration-dependent binding to CHO cells.
[075] [075] The preferred optimized anti-CD47 antibody molecules of the present invention do not necessarily have the maximum number of human germline substitutions in the corresponding murine CDRs or other amino acid positions (such as structure). As elaborated in the experimental section below, we found that antibody molecules "maximally humanized" are not necessarily "maximally optimized" in terms of anti-CD47 binding characteristics and / or other desirable characteristics.
[076] [076] The present invention encompasses modifications to the amino acid sequence of the antibody molecule or its antigen-binding fraction, as defined herein. For example, the invention includes antibody molecules and their corresponding antigen binding parts, comprising functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants that have enhanced or decreased activity or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for CD47. Insertions are envisaged that include amino- and / or carboxyl-terminal fusions of length ranging from one residue to polypeptides containing one hundred or more residues, as well as intrasequent insertions of single or multiple amino acid residues. Examples of terminal inserts include an antibody molecule with an N-terminal methionyl residue or the antibody molecule fused with an epitope tag. Other variants of insertion of the antibody molecule include fusion to the N or C terminal of the antibody of an enzyme or polypeptide that increases the half-life of the antibody in the bloodstream.
[077] [077] The antibody molecule or antigen-binding fraction of the invention can include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation and phosphorylation. The antibody molecule or antigen-binding fraction of the invention can be mutated to alter these post-translational modifications, for example, by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site.
[078] [078] The antibody molecule or antigen-binding fraction of the invention can be modified, for example, by substituting amino acids to remove possible proteolytic sites in the antibody.
[079] [079] In the antibody molecule or its antigen-binding fraction, HCDR1 can have the amino acid sequence: G-Y-T / S / N / R-F-T / N-N / S-Y-Y-I / V-F / V / G (SEQ ID NO: 21); HCDR2 can have the amino acid sequence: M / I-G-V / N / I / D-I-N / Y-P-V / G / F-N / D-G / S-D-T-N / R / K-F / Y-N / S-P-S-F-Q-G (SEQ ID NO: 22); and HCDR3 can have the amino acid sequence: GGF / H / I / Q / YT / V / IM / T / R / P / A / LD / GY / Q / N / R / S / W / K / A / E / F / H / I / L / M / T / V (SEQ ID NO: 23). Alternatively, using the definition of AHo, in the antibody molecule or its antigen-binding fraction, HCDR1 can have the amino acid sequence: G-S-G-Y-T / S / N / R-F-T / N-N / S-Y-Y (SEQ ID NO: 24); HCDR2 can have the amino acid sequence: I- N / Y-P-V / G / F-N / D-G / S-D-T-N / R / K-F / Y-N / S-P-S-F-Q-G (SEQ ID NO: 25); and HCDR3 can have the amino acid sequence: G-G- F / H / I / Q / Y-T / V / I-M / T / R / P / A / L-D / G (SEQ ID NO: 26).
[080] [080] For example, HCDR1 can have the amino acid sequence: G-Y-T / S-F-T-N-Y-Y-I-F (SEQ ID NO: 27); HCDR2 can have the amino acid sequence: M / I-G-I / D-I-N-P-V-N / D-G-D-T-N / R-F / Y-N / S-P-S-F-Q-G (SEQ ID NO: 28); and HCDR3 can have the amino acid sequence: G-G-F / Y-T-M / P-D-Y / R / K / I (SEQ ID NO: 29). Alternatively, using the definition of AHo, HCDR1 can have the amino acid sequence: G-S-G-Y-T / S-F-T-N-Y-Y (SEQ ID NO: 30); HCDR2 can have the amino acid sequence: I-N-P-V-N / D-G-D-T-N / R-F / Y-N / S-P-S-F-Q-G (SEQ ID NO: 31); and HCDR3 can have the amino acid sequence: G-G-F / Y-T-M / P-D (SEQ ID NO: 32).
[081] [081] In the antibody molecule or its antigen-binding fraction, LCDR1 can have the amino acid sequence: RSSQ / HSF / LL / VHSN / Q / AG / AY / N / SN / TY-LH / D (SEQ ID NO: 33); LCDR2 can have the amino acid sequence: L / K / M-V / G-S-N / Y-R-A / F / L / S-S (SEQ ID NO: 34); and LCDR3 can have the amino acid sequence: F / L / M / S / T / VQ- Q / N / A / T / ST / L / M / IQ / HT / V / I / A / FP / LR / WT (SEQ ID NO: 35).
[082] [082] For example, LCDR1 can have the amino acid sequence: R-S-S-Q-S-L-L / V-H-S-N / Q / A-G-Y / N-N / T-Y-L-H / D (SEQ ID NO: 38); LCDR2 can have the amino acid sequence: L / K-V / G-S-N / Y-R-A / F / L-S (SEQ ID NO: 39); and LCDR3 can have the amino acid sequence: F / S-Q-Q / N / A-T / L-Q / H-T / V-P-R-T (SEQ ID NO: 40). Alternatively, using the definition of AHo, LCDR1 can have the amino acid sequence: S-S-Q-S-L-L / V-H-S-N / Q / A-G-Y / N-N / T-Y (SEQ ID NO: 41); LCDR2 can have the amino acid sequence: L / K-V / G-S-N / Y-R-A / F / L-S (SEQ ID NO: 39); and LCDR3 can have the amino acid sequence: Q / N / A-T / L-Q / H-T / V-P-R (SEQ ID NO: 42).
[083] [083] In specific embodiments of the invention, as defined using the unified CDR definition, the antibody molecule or antigen binding fraction may comprise: (a) the GYSFTNYYIF amino acid sequences (SEQ ID NO: 43) (HCDR1) , MGDINPVNGDTNYSPSFQG (SEQ ID NO: 44) (HCDR2), GGYTPDY (SEQ ID NO: 45) (HCDR3), RSSQSLLHSNGYNYLH (SEQ ID
[084] [084] In the specific modalities above of the invention, CDRs can alternatively be defined using the definition of AHo CDR, so that the antibody molecule or antigen-binding fraction comprises: (a) the GSGYSFTNYY amino acid sequences (SEQ ID NO: 86) (HCDR1), INPVNGDTNYSPSFQG (SEQ ID NO: 87) (HCDR2), GGYTPD (SEQ ID NO: 88) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRFS (SEQ ID NO: 47) (LCDR2) and NLHVPR (SEQ ID NO: 90) (LCDR3) [Clone D-H3]; or (b) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5]; or (c) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPVNGDTNFSPSFQG (SEQ ID NO: 94) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLVHSNGYTY (SEQ ID NO: 95))
[085] [085] The antibody molecule or antigen-binding fraction of the invention (defined using the definition of AHo) can comprise:
[086] [086] The above antibody molecule or antigen binding fraction can, alternatively, be defined using the equivalent definitions of Unified CDR, as disclosed here.
[087] [087] In particular embodiments of the invention, the antibody molecule or antigen-binding fraction can comprise the six CDR sequences of Clone A-D5, Clone A-D5.4 or Clone A-D5.16 or Clone A-D5. 16-DI or Clone A-D5-DI as defined above, or a suitable combination of the CDR sequences for each of these clones.
[088] [088] For example, in the antibody molecule or antigen-binding fraction, as defined using the unified CDR definition, HCDR1 can have the amino acid sequence: G-Y-T / S-F-T-N-Y-Y-I-F (SEQ ID NO: 27); HCDR2 can have the amino acid sequence: M-G-I-I-N-P-V-D-G-D-T-N / R-Y-N / S-P-S-F-Q-G (SEQ ID NO: 111); HCDR3 can have the amino acid sequence: G-G-Y-T-M-D-R (SEQ ID NO: 51); LCDR1 can have the amino acid sequence: R-S-S-Q-S-L-H-S-N / A-G-Y-N / T-Y-L-H (SEQ ID NO: 112); LCDR2 can have the amino acid sequence: K-V-S-N-R-L / F-S (SEQ ID NO: 113); and LCDR3 can have the amino acid sequence: F-Q-N-T-H-T-P-R-T (SEQ ID NO: 54).
[089] [089] Alternatively, in the antibody molecule or antigen-binding fraction, as defined using the definition of AHo, HCDR1 can have the amino acid sequence: G-S-G-Y-T / S-F-T-N-Y-Y (SEQ ID NO: 30); HCDR2 can have the amino acid sequence: I-N-P-V-D-G-D-T-N / R-Y- N / S-P-S-F-Q-G (SEQ ID NO: 114); HCDR3 can have the amino acid sequence: G-G-Y-T-M-D (SEQ ID NO: 16); LCDR1 can have the amino acid sequence: S-S-Q-S-L-L-H-S-N / A-G-Y-N / T-Y (SEQ ID NO: 115); LCDR2 can have the amino acid sequence: K-V-S-N-R-L / F-S (SEQ ID NO: 113); and LCDR3 can have the amino acid sequence: N-T-H-T-P-R (SEQ ID NO: 93).
[090] [090] The antibody molecule or antigen-binding fraction, as defined herein, may comprise one or more substitutions, deletions and / or insertions that remove a post-translational modification (PTM) site, for example, a glycosylation site (linked to N or linked to O), a deamination site, a phosphorylation site or an isomerization / fragmentation site.
[091] [091] More than 350 types of PTM are known. The main forms of PTM include phosphorylation, glycosylation (linked to N and O), soil, palmitoylation, acetylation, sulfation, myristoylation, pre-alkylation and methylation (of K and R residues). Statistical methods for identifying putative amino acid sites responsible for specific PTMs are well known in the art (see Zhou et al., 2016, Nature Protocols 1: 1318-1321). The removal of such a site, for example, by substitution, exclusion and / or insertion and then optional testing (experimentally and / or theoretically) for (a) binding activity and / or (b) loss of PTM is contemplated.
[092] [092] For example, murine LCDR1 VxP037 (as defined herein, that is, the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9)) has been identified as having a putative deamidation site at residue 10 (N) and / or 12 (N). The removal of either of these two locations in equivalent positions on an LCDR1 of the invention, for example, by conservative substitution (such as S, A, Q or D), is foreseen (as for example in clone A-D5.8, clone A -D5 and other clones in Tables 3 and 4, or clones A-D5.11 to A-D5.18 in Table 5).
[093] [093] Likewise, murine LCDx3 VxP037 (as defined herein, that is, the amino acid sequence SQNTHVPRT (SEQ ID NO: 11)) was identified as having a putative deamidation site at residue 3 (N). The removal of this site in an equivalent position on an LCDR3 of the invention, for example, by conservative or non-conservative substitution (such as A, S, H, D, T, K, G, E, Q or R), is provided (as for example in clone F-E7 and other clones in Tables 3 and 4).
[094] [094] Likewise, the murine HCDR3 VxP037 (as defined herein, that is, the amino acid sequence GGYTMDY (SEQ ID NO: 5)) was identified as having a putative oxidation site at residue 5 (M). Removal of this site in an equivalent position on an HCDR3 of the invention, for example, by conservative or non-conservative substitution (such as P, A, T, S, L, F, W, V, I, Y or R), is provided ( as for example in clone D-H3 and other clones in Tables 3 and 4).
[095] [095] The antibody molecule or its antigen-binding fraction can be human, humanized or chimeric.
[096] [096] The antibody molecule or its antigen-binding fraction may comprise one or more human variable domain structure supports into which the CDRs have been inserted.
[097] [097] The antibody molecule or its antigen-binding fraction may comprise a human germline support IGHV5-51 in which the corresponding HCDR sequences have been inserted.
[098] [098] The antibody molecule or its antigen-binding fraction may comprise a human germline support IGKV2-28 in which the corresponding LCDR sequences have been inserted.
[099] [099] The antibody molecule or its antigen-binding fraction may comprise an immunologically inert constant region.
[100] [100] The antibody molecule or its antigen-binding fraction can be a Fab fragment, an F (ab) 2 fragment, an Fv fragment, a tetrameric antibody, a tetravalent antibody, a multispecific antibody (for example, a bivalent antibody ), a single antibody domain (for example, a shark antibody [VNAR antibody], or a fragment thereof, or a camelid antibody [VHH antibody], or a fragment thereof), a monoclonal antibody, or a fusion protein. Antibody molecules and methods for their construction and use are described, for example, Holliger & Hudson (2005, Nature Biotechnol. 23 (9): 1126-1136).
[101] [101] In another aspect of the invention, an immunoconjugate is provided comprising the antibody molecule or its antigen-binding fraction of the invention, as defined herein, attached to a therapeutic agent.
[102] [102] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulating agents, antiangiogenic agents, antiproliferative agents, pro-apoptotic agents and cytostatic and cytolytic enzymes (for example RNAs). Other therapeutic agents include a therapeutic nucleic acid, such as a gene that encodes an immunomodulatory agent, an antiangiogenic agent, an antiproliferative agent or a pro-apoptotic agent. These drug descriptors are not mutually exclusive and, therefore, a therapeutic agent can be described using one or more of the above terms.
[103] [103] Examples of therapeutic agents suitable for use in immunoconjugates include taxanes, maytansines, CC-1065 and duocarmicins, calicheamicins and other enedins and auristatins. Other examples include anti-folates, vinca alkaloids and anthracyclines. Plant toxins, other bioactive proteins, enzymes (ie, ADEPT), radioisotopes, photosensitizers can also be used in immunoconjugates. In addition, conjugates can be made using secondary vehicles as a cytotoxic agent, such as liposomes or polymers. Suitable cytotoxins include an agent that inhibits or prevents cell function and / or results in cell destruction. Representative cytotoxins include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind and interrupt DNA and agents that interrupt protein synthesis or the function of essential cellular proteins, such as protein kinases, phosphatases, topoisomerases, enzymes and cyclins.
[104] [104] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaryl, pitarubicin, valrubicin, cytarabine, gemcitabine, enzyme , pentostatin, broxuhdina, capecitabine, cladhbina, decitabine, floxuhdina, fludarabine, gougerotine, puromycin, tegafur, tiazofuhn, adamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, proclastamine, proclastamine, vinctristine, vinctristine , etoposide, taxol, taxol analogues, platinum such as cis-platinum and carbo-platinum, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatines / auristatins, hemiasterins, hemiasterines and maytansinoids.
[105] [105] Suitable immunomodulatory agents include anti-hormones that block hormonal action in tumors and immunosuppressive agents that suppress cytokine production, negatively regulate self-antigen expression or mask MHC antigens.
[106] [106] Also provided is a nucleic acid molecule that encodes the antibody molecule or its antigen-binding fraction of the invention, as defined herein.
[107] [107] A vector is further provided comprising the nucleic acid molecule of the invention, as defined herein.
[108] [108] A host cell comprising the nucleic acid molecule or vector of the invention as defined herein is also provided.
[109] [109] In a further aspect, a method is provided to produce an anti-CD47 antibody and / or an antigen binding fraction, comprising culturing the host cell of the invention under conditions that result in the expression and / or production of the antibody and / or its antigen-binding fraction and antibody isolation and / or its antigen-binding fraction from the host cell or culture.
[110] [110] In another aspect of the invention, a pharmaceutical composition is provided comprising the antibody molecule or its antigen-binding fraction of the invention, as defined herein, or the nucleic acid molecule of the invention, as defined herein, or the vector of the invention, as defined herein.
[111] [111] In addition, a method is provided to improve an immune response in an individual, comprising administering an effective amount of the antibody molecule or its antigen-binding fraction as defined herein, or the immunoconjugate of the invention, as defined herein, either the nucleic acid molecule of the invention as defined herein, or the vector of the invention as defined herein, or the pharmaceutical composition of the invention as defined herein.
[112] [112] In a further aspect, a method is provided to treat or prevent cancer in an individual, comprising administering an effective amount of the antibody molecule or its antigen-binding fraction as defined herein, or the immunoconjugate of the invention, as defined herein, either the nucleic acid molecule of the invention as defined herein, or the vector of the invention as defined herein, or the pharmaceutical composition of the invention as defined herein.
[113] [113] Cancer can, for example, be selected from the group consisting of: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, cancer bile ducts, cancer of the small intestine or appendix, cancer of the salivary gland, cancer of the thyroid gland, cancer of the adrenal gland, osteosarcoma, chondrosarcoma and cancer of hematological tissues.
[114] [114] The invention also provides an antibody molecule or its binding fraction to the antigen of the invention, as defined herein, or the immunoconjugate of the invention, as defined herein, or the nucleic acid molecule of the invention, as defined herein, or the vector of the invention, as defined herein, or the pharmaceutical composition of the invention, as defined herein, for use in the treatment of cancer.
[115] [115] In another aspect, the invention provides the antibody molecule, or its antigen-binding fraction, or the immunoconjugate, or the nucleic acid molecule, or the vector for use, or the method of treatment of the invention, as defined here, for separations, sequential or sequential.
[116] [116] In a further aspect, use is made of an antibody molecule or antigen-binding fraction of the invention, as defined herein, or an immunoconjugate of the invention, as defined herein, or a nucleic acid molecule of the invention, as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the manufacture of a medicament for treating cancer.
[117] [117] The invention also provides a method for treating or preventing an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease in an individual, comprising administering an effective amount of the antibody molecule or its fraction of binding to the antigen, as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein.
[118] [118] Ischemia-reperfusion injury in all aspects can occur in organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resections and reconstructive surgery, replacement from an appendix or other body part, skin graft or trauma.
[119] [119] Autoimmune or inflammatory disease can be selected from the group consisting of: arthritis, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave's disease and Hashimoto's thyroiditis and ankylosing spondylitis.
[120] [120] An antibody molecule or antigen-binding fraction as defined herein is also provided, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein, for use in the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
[121] [121] In addition, use is made of an antibody molecule or its antigen-binding fraction, as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein , or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
[122] [122] The invention also provides a method for the treatment or prevention of cardiovascular disease or fibrotic disease in an individual, comprising administering an effective amount of the antibody molecule or its antigen-binding fraction, as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein.
[123] [123] An antibody molecule or antigen-binding fraction as defined herein is also provided, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein, for use in the treatment of cardiovascular disease or fibrotic disease.
[124] [124] In addition, use is made of an antibody molecule or its antigen-binding fraction, as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein , or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of cardiovascular disease or fibrotic disease.
[125] [125] Cardiovascular disease in any aspect of the invention can be for example coronary heart disease or atherosclerosis.
[126] [126] Fibrotic disease in any aspect of the invention can be selected from the group consisting of myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis and bronchitis.
[127] [127] The pharmaceutical composition of the invention can comprise a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can be a compound or a combination of compounds that enter a pharmaceutical composition that does not cause side reactions and that allows, for example, to facilitate the administration of the anti-CD47 antibody molecule, an increase in its useful life and / or its effectiveness in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art depending on the mode of administration of the anti-CD47 antibody molecule.
[128] [128] In some embodiments, the anti-CD47 antibody molecule can be supplied in a lyophilized form for reconstitution before administration. For example, lyophilized antibody molecules can be reconstituted in sterile water and mixed with saline prior to administration to an individual.
[129] [129] Anti-CD47 antibody molecules will normally be administered in the form of a pharmaceutical composition, which can comprise at least one component in addition to the antibody molecule. Thus, the pharmaceutical compositions may comprise, in addition to the anti-CD47 antibody molecule, an excipient, vehicle, buffer, stabilizer or other pharmaceutically acceptable materials well known to those skilled in the art.
[130] [130] For parenteral administration, for example, subcutaneous or intravenous administration, for example, by injection, the pharmaceutical composition comprising the anti-CD47 antibody molecule can be in the form of a parenterally acceptable aqueous solution that is pyrogen free and has a pH adequate isotonicity and stability. Those skilled in the art are able to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride injection, Ringe injection, lactated Ringe injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be used as needed, including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; paraben alcohol, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine,
[131] [131] A pharmaceutical composition comprising an anti-CD47 antibody molecule can be administered alone or in combination with other treatments, dependent simultaneously or sequentially on the condition to be treated.
[132] [132] An anti-CD47 antibody molecule, as described here, can be used in a method of treating the human or animal body, including prophylactic or preventive treatment (for example, treatment before the start of a condition in an individual to reduce the risk of the condition occurring in an individual; delaying its onset or reducing its severity after onset). The treatment method may comprise administering the anti-CD47 antibody molecule to an individual in need thereof.
[133] [133] Administration is usually in a "therapeutically effective amount", being sufficient to show benefit to a patient. This benefit can be at least the improvement of at least one symptom. The actual amount administered, the rate and time of administration will depend on the nature and severity of what is being treated, the specific mammal being treated, the clinical condition of each patient, the cause of the disorder, the place of treatment. delivery of the composition, the method of administration, the schedule of administration and other factors known to doctors.
[134] [134] A typical dose of antibody will be in the range of 100 µg to 1 g for systemic applications and 1 µg to 1 mg for topical applications. A higher starting dose may be administered, followed by one or more lower doses.
[135] [135] Treatment can be periodic and the period between administrations can be about two weeks or more, for example, about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment can be every two to four weeks or every four to eight weeks. Treatment can be administered before and / or after surgery and / or can be administered or applied directly to the anatomical site of the surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.
[136] [136] In some embodiments, anti-CD47 antibody molecules, as described herein, can be administered as subcutaneous injections. Subcutaneous injections can be administered using an auto injector, for example, for long-term prophylaxis / treatment.
[137] [137] In some preferred embodiments, the therapeutic effect of the anti-CD47 antibody molecule may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of the anti-CD47 antibody molecule may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more or 6 months or more.
[138] [138] The invention also provides a method for producing an antibody molecule that specifically binds to human CD47 and, optionally, also to cynomolgus monkey CD47 and / or mouse CD47, or an antigen-binding fraction, comprising the steps of: (1) grafting anti-CD47 CDRs from a non-human source into a human v-domain structure to produce a humanized anti-CD47 antibody molecule or antigen binding fraction thereof; (2) generate a phage library of clones of the humanized anti-CD47 antibody molecule or its antigen-binding fraction, comprising one or more mutations in the CDRs; (3) screening the phage library for binding to human CD47 and optionally also to cynomolgus monkey CD47 and / or mouse CD47; (4) selecting clones from the screening step (3) having specificity of binding to human CD47 and optionally also to CD47 of cynomolgus monkey and / or mouse CD47; and (5) production of an antibody molecule that specifically binds to human CD47 and, optionally, also to CD47 of the cynomolgus monkey and / or mouse CD47, or to an antigen-binding fraction of the clones selected in step (4 ).
[139] [139] The method may comprise an additional step of producing additional clones based on the clones selected in step (4), for example, based on additional exploratory mutagenesis at specific positions in the CDRs of the clones selected in step (4), for improve humanization, minimize content of the human T cell epitope and / or improve the manufacturing properties in the antibody molecule or its antigen binding fraction produced in step (5).
[140] [140] The method may comprise an additional step of assessing the immunogenicity of one or more v domains in the clones selected in step (4) or in the antibody molecule produced in step (5) and, optionally, generating one or more additional mutations, for example, in a CDR and region of the structure, to reduce immunogenicity. Immunogenicity can be assessed by identifying the location of T cell epitopes, for example, using in silico technologies as described herein.
[141] [141] The refinements applicable to the above method are those described in Example 1 below.
[142] [142] As used here, the term "CD47" refers to Integrin-Associated Protein (IAP) and its variants that retain at least part of the biological activity of CD47. As used herein, CD47 includes all mammalian species of the native CD47 sequence, including humans, rats, mice and chickens. The term "CD47" is used to include variants, isoforms and homologs of human CD47 species.
[143] [143] As used herein, an "antagonist" as used in the context of the antibody of the invention or an "anti-CD47 antagonist antibody" (termed "anti-CD47 antibody") refers to an antibody capable of binding to CD47 and inhibiting the biological activity of CD47 and / or downstream pathways mediated by CD47 signaling. An antagonist anti-CD47 antibody comprises antibodies that can block, antagonize, suppress or reduce (including significantly) the biological activity of CD47, including downstream pathways mediated by CD47 signaling, such as receptor binding and / or obtaining a cellular response to CD47. For the purposes of the present invention, it will be explicitly understood that the term "anti-CD47 antagonist antibody" encompasses all terms, titles and functional states and characteristics by which the biological activity of CD47 and CD47 itself (including, but not limited to, its ability to increase phagocytosis activation by cells of the myeloid lineage), or the consequences of biological activity or activity, are substantially nullified, diminished, or neutralized to any significant degree.
[144] [144] CD47 "specifically binds" "specifically interacts", "preferentially binds", "binds" or "interacts" with CD47 if it binds with greater affinity,
[145] [145] An "antibody molecule" is an immunoglobulin molecule capable of specific binding to a target, such as carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the molecule immunoglobulin.
[146] [146] An "antibody molecule" comprises an antibody of any class, such as IgG, IgA or IgM (or its subclass), and the antibody need not be of any specific class.
[147] [147] The term "antigen-binding fraction" of an antibody molecule, as used herein, refers to one or more fragments of an intact antibody that retains the ability to specifically bind to CD47. The antigen-binding functions of an antibody molecule can be performed by fragments of an intact antibody. Examples of binding fragments included in the term "antigen binding fraction" of an antibody molecule include Fab; Fab '; F (ab ') 2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody fragment (dAb) and an isolated complementarity determining region (CDR).
[148] [148] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” can be a native sequence Fc region or a region
[149] [149] An "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As known in the art, the variable regions of the heavy and light chain consist of four structural regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions, contributing to the formation of the antibody antigen binding site. When choosing FR to flank CDRs, for example, when humanizing or optimizing an antibody, FRs of antibodies that contain CDR sequences in the same canonical class are preferred.
[150] [150] The CDR definitions used in this application combine the domains used in the many disparate and often conflicting schemes that have been created in the field,
[151] [151] Table 1 shows the amino acid sequences of the CDRs of the murine anti-CD47 antibody VxP037, as defined here (a "Unified" scheme), compared to well-known alternative systems for defining the same CDRs.
[152] [152] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid that does not significantly alter functional activity. A preferred example of a "conservative substitution" is the replacement of an amino acid with another amino acid that has a value ≥ 0 in the following BLOSUM 62 substitution matrix (see Henikoff and Henikoff, 1992, PNAS 89: 10915-10919):
[153] [153] The term "monoclonal antibody" (Mab) refers to an antibody, or its antigen-binding fraction, which is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced. Preferably, a monoclonal antibody of the invention exists in a homogeneous or substantially homogeneous population.
[154] [154] A "humanized" antibody molecule refers to a form of non-human antibody molecules (for example, murine), or their antigen-binding fraction, which are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other substrates of antigen binding antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies can be human immunoglobulins (recipient antibody) in which the residues of a CDR of the recipient are replaced by residues of a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit with the specificity, desired affinity and ability .
[155] [155] "Human antibody or fully human antibody" refers to an antibody molecule, or its antigen-binding fraction, derived from transgenic mice that carry genes from human antibodies or from human cells.
[156] [156] The term "chimeric antibody" is intended to refer to an antibody molecule, or its antigen-binding fraction, in which variable region sequences are derived from one species and constant region sequences are derived from another species, as an antibody molecule whose variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
[157] [157] "Antibody-drug conjugate" and "immunoconjugate" refer to an antibody molecule, or its antigen-binding fraction, including derivatives of antibodies that bind to CD47 and are conjugated to cytotoxic, cytostatic and / or therapeutic agents .
[158] [158] The antibody molecules of the invention, or their antigen-binding fraction, can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art.
[159] [159] The term "epitope" refers to the portion of a molecule capable of being recognized and bound by an antibody molecule or its antigen-binding fraction, in one or more of the antigen-binding regions of the antibody molecule. Epitopes can consist of defined regions of the secondary or tertiary primary protein structure and include combinations of secondary structural units or structural domains of the target recognized by the antigen-binding regions of the antibody or its antigen-binding fraction. Epitopes can also consist of a defined chemically active surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term "antigenic epitope", as used here, is defined as a portion of a polypeptide to which an antibody molecule can specifically bind, as determined by any method well known in the art, for example, by conventional immunoassays, binding assays competitive to antibodies or by X-ray crystallography or related structural determination methods (eg NMR).
[160] [160] The term "binding affinity" or "KD" refers to the rate of dissociation of a particular antigen-antibody interaction. The KD is the ratio of the decoupling rate, also called "off-rate (koff)", to the association rate or "on-rate (kon)". Thus, KD is equal to koff / kon and is expressed as a molar concentration (M). It follows that the lower the KD, the greater the bond affinity. Therefore, a KD of 1 µΜ indicates poor binding affinity compared to a KD of 1 nM.
[161] [161] The term "potency" is a measure of biological activity and can be designated as an IC50, or effective concentration of an antibody or antibody conjugated to the CD47 antigen to inhibit 50% of the activity measured in a CD47 activity assay as described here.
[162] [162] The phrase "effective amount" or "therapeutically effective amount", as used herein, refers to an amount needed (in dosages and for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount is at least the minimum amount, but less than a toxic amount, of an active agent that is necessary to confer therapeutic benefit to an individual.
[163] [163] The term "inhibit" or "neutralize", as used herein in relation to the bioactivity of an antibody molecule of the invention, means the ability of the antibody to antagonize, prohibit, prevent, restrict, delay, substantially slow down, slow down, stop , substantially slow down, eliminate, stop, reduce or reverse eg progression or severity of what is being inhibited, including, but not limited to, a biological activity or binding interaction of the antibody molecule to CD47.
[164] [164] A "host cell" includes an individual cell or cell culture that can or has been a recipient of vectors for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in the complement of genomic DNA) to the original cell, due to natural, accidental or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide (s) of this invention.
[165] [165] As used here, "vector" means a construct that is capable of delivering and, preferably, expressing one or more gene (s) or sequence (s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA expression vectors or RNA encapsulated in liposomes and certain eukaryotic cells, such as producer cells.
[166] [166] The term "treatment", as used here, unless otherwise indicated, means to reverse, alleviate, inhibit the progress of, delay progression, delay the onset or prevent the disorder or condition to which that term applies , or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as defined above. The term "treatment" also includes adjuvant and neoadjuvant treatment for an individual. For the avoidance of doubt, the reference here to "treatment" includes reference to curative, palliative and prophylactic treatment. For the avoidance of doubt, references here to "treatment" also include references to curative, palliative and prophylactic treatment.
[167] [167] It is understood that, whenever embodiments are described here with the language “comprising”, otherwise similar embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.
[168] [168] Where aspects or modalities of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the group main group, but also the main group is absent from one or more group members. The present invention also provides for the explicit exclusion of one or more of any of the group members in the claimed invention.
[169] [169] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which this invention belongs. In case of conflict, this specification, including definitions, will prevail.
[170] [170] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
[171] [171] Particular non-limiting modalities of the present invention will now be described with reference to the accompanying drawings.
[172] [172] In this example, we have successfully generated a panel of optimized and antagonistic anti-CD47 antibodies. These anti-CD47 antibodies are well expressed, biophysically stable, highly soluble and have identity maximized to preferred human germ lines.
[173] [173] The DNA sequences encoding the antibody's v domain were cloned via link-restriction cloning into separate heavy and light chain IgG expression cassettes into separate plasmid vectors. The antibodies were expressed in two forms of manipulated human IgG: IgG4 with the S228P mutation to stabilize the IgG4 hinge and IgG1null - IgG1 with the lower hinge L234A / L235A / G237A mutations, which minimize the effector functions triggered by the Fcγ receptor. IgGs were expressed in HEK-293pi cells after transient transfection with endotoxin-free IgG expression plasmid preparations, according to the manufacturer's protocols. IgGs were purified using a single step protocol: The conditioned medium was loaded (pure) onto a 1 ml ProA sepharose column, pre-equilibrated in PBS pH 7.4. The column was washed with 5 column volumes of PBS pH 7.4, before the protein was eluted with 100 mM glycine, pH 2.7 and subjected to dialysis in PBS pH 7.4 using a 30 kDa cut dialysis membrane.
[174] [174] To coat the Greiner Bio-One High high-binding ELISA plates, the target proteins were diluted to 1 µg / ml in carbonate buffer and added to 100 µl per well at 4 ° C, o / n. The coated plates were washed 3x with PBS pH 7.4, blocked with 1% BSA in PBS (380 µl / well) for 1 hour at room temperature, then washed 3x with PBS-Tween 20 (PBST). CD47 antibodies (100 µl / well; diluted in PBST) were then added and then incubated 1 hour at room temperature. The plates were then washed 3x with PBST and HRP of the goat anti-human kappa chain (100 µl / well) was added to the RT for 1 hour. The plates were then washed 3x with PBST and twice with PBS before adding 100 µL of TMB per well.
[175] [175] Anti-CD47 antibodies were tested for polyreactivity by ELISA. Purified, recombinant, target and non-target antigens were coated on 96-well Nunc maxisorp plates at 100 ng per well in carbonate buffer, at 4 ° C overnight. The plates were then washed 3x with PBS, blocked with 1% BSA in PBS, then washed 3x with PBS-Tween20. A series of dilutions of primary antibodies was then applied, the plates were washed 3x with PBS-Tween20 followed by application of secondary human kappa chain anti-goat-HRP antibody 1: 4,000.
[176] [176] The CD47 scFv repertoire was assembled by mass synthesis of oligo and PCR. The amplified scFv repertoire was then cloned via restriction ligation into a phage vector, transformed into E.coli TG-1 cells, and the phage repertoire rescued essentially as previously described in detail (Finlay et al., 2011, Methods Mol Biol 681 : 383- 401).
[177] [177] Phage selections were performed by coating magnetic streptavidin microspheres with CD47-Fc protein (human or chino), washing the beads three times with PBS and resuspending them in PBS pH 7.4 plus skim milk protein 5 % (MPBS). These beads were coated with a 200 nM target protein in round 1 of selection, followed by 100, 50 and 10 nM in subsequent rounds.
[178] [178] A competition ELISA assay was established to examine the ability of derivations optimized to block the binding interaction of CD47 with SIRPα. To coat Greiner Bio-One High bind ELISA plates, 10 µg / ml of human SIRPα-Fc in carbonate coating buffer was added to 100 µl per well at 4 ° C, o / n. The coated plates were washed 3x with PBS pH 7.4, blocked with 1% BSA in PBS (380 µl / well) for 1 hour at room temperature, then washed 3x with PBS-Tween 20 (PBST). CD47- Human Fc, mouse or biotinylated cino was then added to 0.2 µg / ml in PBS, 100 µl per well, at room temperature for 60 minutes with or without the addition of competing IgGs. The plates were then washed 3x with PBST and added Streptavidin-HRP (100 µl / well) at room temperature for 1 hour. The plates were then washed 3x with PBST and twice with PBS before adding 100 µL of TMB per well. The reactions were stopped by adding 100 µL of 2M H2SO4 / well and the OD was read on a plate reader at 450 nm.
[179] [179] In silico technologies (Abzena, Ltd.), which are based on identifying the location of T cell epitopes in therapeutic antibodies and proteins, have been used to assess the potential immunogenicity in the v domains of the antibody. ITopeTM was used to analyze the VL and VH sequences of the main leads for peptides with high-affinity promiscuous binding to human class II MHC.
[180] [180] In addition, the sequences were analyzed using the TCEDTM (T Cell Epitope DatabaseTM) search to find matches with T cell epitopes previously identified by in vitro human T cell epitope mapping analyzes of other protein sequences. TCEDTM is used to search any test sequence in a large database (> 10,000 peptides) of peptides derived from unrelated proteins and antibody sequences.
[181] [181] Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation. The CD14 + PBMCs were subsequently isolated by isolating magnetic cells using CD14 microspheres. In parallel, HL-60 cells were labeled using a CFSE green cell tracer dye (carboxy-fluorescein diacetate, succinimidyl ester). A total of 1.25 x 106 labeled HL-60 cells were pre-incubated in the presence of anti-CD47 antibodies in 24-well plates for 1 hour at 37 ° C in a humidified atmosphere containing 5% CO2. After incubation, 5 x 105 CD14 positive cells were added to each well and incubated for another hour under the same culture conditions. The cells were harvested by vigorous pipetting, fixed using cold 4% paraformaldehyde for 10 minutes and then blocked with a monoclonal antibody inhibiting binding to the Fc receptor for 10 minutes. After the blocking step, the cells were incubated with an anti-human CD14 antibody conjugated to Alexa Fluor 647 (AF647) at room temperature for 30 minutes and set an additional time in 4% paraformaldehyde for 5 minutes.
[182] [182] The cells were analyzed on the BD Fortessa flow cytometer, recording the lateral dispersion and direct dispersion properties, together with CFSE and AF647 fluorescence intensity data. The data was captured until at least 1 x 104 positive AF647 events were recorded. The data were analyzed post-acquisition using FlowJo software (version 10.4.2).
[183] [183] The CDRs of an antagonistic anti-CD47 IgG murine VP037 (Mvh / MVL; see WO2014 / 093678 and in Table 2) were initially introduced into the germline structural immunoglobulin V-domain human sequence scaffolds using CDR grafting. To polarize our engineering efforts to provide definitive therapeutic IgG compounds with optimal drug-like properties, we opted for grafting CDRs from the parental antibody to “preferred” germline scaffolds IGHV5-51 and IGKV2-28, which are known for have good solubility and are used with high frequency in the expressed human antibody repertoire.
[184] [184] These grafted CDR structures and definitions are described in Table 2. The heavy and light chain sequences for murine anti-CD47 antibody are also shown in Table 2. Although this CDR grafting process is well known, it is still problematic to predict whether a given set of human v domain sequences will act as suitable acceptor structures for non-human CDR grafting. The use of inappropriate structures can lead to loss of the target binding function, protein stability problems or even impaired expression of the final IgG. The IGHV5-51 / IGKV2-28 graft was therefore taken forward as a model for CDR mutagenesis and selection of improved clones.
[185] [185] Sequences of the v IGHV5-51 / IGKV2-28 domain grafted into CDR were combined into a VL-VH scFv format and a cassette from the mutagenesis library was generated by mass oligo synthesis and assembly. The final scFv library was linked to a phage display vector and transformed into E. coli by electroporation to generate 1.3 x 109 independent clones. The construction quality of the library was verified by sequencing 96 clones. These sequencing data showed that the positions encoding the murine or human germline residue in each variation position were effectively sampled at a frequency of approximately 50%. The libraries were rescued using the auxiliary phage M13 and selections were made on the biotinylated CD47-Fc proteins of humans, mice and cynomolgus monkeys in three separate branches A, B and C.
[186] [186] Post-selection screening (Fig. 1) and DNA sequencing revealed the presence of 854 unique scFv clones, binding to human and mouse CD47, with significantly increased human content in CDRs, while structural sequences remained fully in the germ line. Among these 854 clones, mutations in the germ lining were observed in all CDRs (Table 3). The main clones were classified based on the level of CDR germ-coat against the ELISA signal for binding to both human and mouse CD47-Fc (Fig.
[187] [187] Although mutations in the germ lining have been observed in all CDRs for the main clones derived directly from library selections, it remained possible that sequence analyzes would allow other clones to be designed for maximum humanization. The 854 unique successes in the sequence with binding signals against human and mouse protein were therefore used to analyze the frequency of murine amino acid retention in the CDRs of this functionally characterized population. The frequency of positional amino acid retention was expressed as a percentage found in the VH and VL domains (Fig. 2A and B). Murine residues with RF <75% were considered to be positions that are possibly not essential for the target binding parotope and are probably open to germination alignment in a series of combinatorial projects.
[188] [188] A project containing only murine residues with RF> 75% was designated "MH" (MH = Maximamente Humanizado).
[189] [189] The purified IgGs described above were then tested for binding to human, mouse and cino CD47-Fc in the direct titration ELISA format (Fig. 3).
[190] [190] In a CD47-SIRPα blocking assay (Fig. 4), A-D5, G-B6, F-E7 and D-H3 exhibited all concentration-dependent blocks of human, mouse and cino CD47 interaction with SIRPα-Fc The concentration varies as the mVH / mVL IgG1 antibody. Notably, VH-A1 / VL-B1 IgG1 exhibited potent human and cyano CD47 block, but no mouse CD47 block. Interestingly, the MH clone, despite having demonstrated binding to all 3 CD47 orthologists in the ELISA, showed no sign of blockage in any assay. The TTP clone was also negative in all blocking assays.
[191] [191] To ensure that the main clones have not suffered loss of target specificity during the mutation and re-selection process; the main and control IgG1 clones were tested for binding to a panel of 14 purified human proteins from the immunoglobulin superfamily (Fig. 5). All five IgG showed signs of binding at 1 µg / ml on CD47-Fc (human OD450 nm> 2.0, cino> 2.0, mouse> 1.25) and no detectable binding (OD450 nm <0.1) against any other protein. A notable exception was the VH-A1 / Vl-B1 clone, which again showed a strong link to human CD47 and cytoplasm, but no signal against the mouse CD47.
[192] [192] Antibodies to CD47 were analyzed for concentration-dependent binding to the cell surface using flow cytometry. CHO-K1 cells were stably transfected with human, mouse CD47 or full-length cino cDNAs. The anti-CD47 IgG mVH / mVL, VH-A1 / VL-B1, A-D5, G-B6, F-E7 and D-H3 and an IgG1 isotype control were all tested in the IgG1null and IgG4 (S228P) formats, next to a monoclonal anti-human CD47 anti-human MS47 CD1, above a concentration range of
[193] [193] To examine the binding of major IgGs to human CD47 + cancer cells, HL60 cells (derived from human acute myeloid leukemia) were also used in flow cytometry analyzes, as above.
[194] [194] It is known in the art that the binding of IgGs intended for therapeutic use to various indicative biological substrates is an indicator of high risk of poor performance in patients due to low bioavailability and short half-life in vivo.
[195] [195] As described above, clone A-D5 proved to have highly specific binding to human, cyano and mouse CD47, low off-target binding potential, improved neutralization of mouse CD47, reduced background binding to CHO cells and multiple human germline mutations in CDRs. As a library-derived clone, however, the A-D5 sequence still retained a number of non-germinative residues (mouse derivatives) that were suggested as potentially superfluous by the data found in Figs 2A and 2B. The A-D5 sequence and all other library-derived clones also maintained a 'NG' motif on the CDR-L1 that presented a high risk of deamidation, as it was found to exhibit high exposure to solvents at the apex of the long and flexible IGKV2 -28 loop Germline model CDR-L1.
[196] [196] In the second phase, a series of other mutants were designed on the best performing mutant in phase 1: A-D5.4
[197] [197] Derivatives A-D5 A-D5.4 and A-D5.16 were subsequently also tested in IgG1null format to maintain binding specificity to ensure that there was no loss of target specificity during the mutation and re-selection process ; both clones were tested for binding to a panel of 14 purified human proteins from the immunoglobulin superfamily (Fig. 13).
[198] [198] Finally, mutants of the A-D5 derivative A-D5.4 and A-D5.16 were examined for their binding to CHO cells of the wild type (Fig. 15) and HL60 (Fig. 16) by cytometry of flow. These analyzes confirmed that the original murine v domains of the mVH / mVL clone in IgG1null or
[199] [199] In silico technologies (Abzena, Ltd.), which are based on identifying the location of T cell epitopes in therapeutic antibodies and proteins, have been used to assess the immunogenicity of the v mVH / mVL and main antibody domains. The analysis of domain v sequences was performed with overlapping 9mer peptides (with each overlapping the last peptide by 8 residues) that were tested against each of the 34 MHC class II allotypes.
[200] [200] Peptides were grouped into four classes: High Foreign Affinity ('HAF' - high risk of immunogenicity), Low Foreign Affinity ('LAF' - lowest risk of immunogenicity), TCED + (epitope previously identified in the TCEDTM database) and Germ Line Epitope ('GE' - peptide sequence of the human germ line with high affinity for binding to MHC Class II). Germline 9mer epitope peptides are unlikely to have immunogenic potential due to T cell tolerance (ie, these peptides are recognized as 'self' in the host), as validated by previous studies with a wide range of germline peptides . It is important to note that these epitopes of the germline v domain (aided by similar sequences in the constant regions of human antibodies) also compete for the occupation of MHC Class II in the membrane of antigen presenting cells, reducing the risk of presenting foreign peptides being sufficient to achieve the 'threshold' activation necessary for stimulation of T cells. a high GE content is therefore a beneficial quality in the clinical development of a therapeutic antibody.
[201] [201] As shown in Figure 17, the major v major domains exhibited significant beneficial changes in the content of the peptide epitope compared to mVH / mVL.
[202] [202] The above findings enabled the design of a maximally de-immunized light chain sequence that was paired with the A-D5.16 VH sequence (Table 4) to form the 'A-D5.16-DI' clone. A-D5.16-DI contained the LCDR sequence 'RSSQSLLHSAGYNYLH' (SEQ ID NO: 82), the LCDR2 sequence 'KVSNRFS' (SEQ ID NO: 85) and the 2-LCDR3 structure sequence 'AGVYYCFQNTHTPRT' (SEQ ID NO : 122) (residues in Table 2 underlined). This clone was readily expressed in the IgG1null format and was found to retain the target binding affinity against human, cyano and mouse CD47 (Fig. 18. A-C) comparable to the mVH / mVL IgG (reduced in Fig.
[203] [203] Studies have been carried out to examine the relative potency of CD47 blockade in the conduction of HL60 human cancer cell phagocytosis by primary human macrophages. As shown in Fig. 20A, in the format of IgG4 (S228P) - mVH / mVL, A-D5, A-D5.4 and A-D5.16, all conducted significant phagocytosis at all concentrations tested.
[204] [204] Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations to them can be made without departing from the spirit and scope of the present invention and that such modifications are clearly contemplated here. No limitation with respect to the specific modalities disclosed in this document and set out in the appended claims is intended or should be inferred.
[205] [205] All documents cited herein are incorporated by reference in their entirety.
Table 1. Amino acid sequences of murine anti-CD47 CDRs, as defined here ("Unified" scheme) compared to alternative definitions. SEQ ID NOs are shown in parentheses.
Scheme HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Unified GYTFTNYYVF IGDINPVNGD GGYTMDY LIYKVSYRFS SQNTHVPRT (4) TNFNEKFKN (5) RSSQSLVHSNG (10) (11) (123) NTYLHW (9) YY Kabat GYTFTNYYVF IGDINPVNGD TRGGYTMDY LIYKVSYRFS SQNTHVPRT (124) TNFNEKFKN (5) RSSQSLVHSNG ( 10) (11) (125) NTYLHW (9) Y Chotia GYTFTNYYVF IGDINPVNGD TRGGYTMDY RSSQSLVHSNG LIYKVSYRFS SQNTHVPRT (126) TNFNEKFKN (5) NTYLHW (10) (11) (127) (9)
IMGT GYTFTNYYVF IGDINPVNGD TRGGYTMDY RSSQSLVHSNG LIYKVSYRFS SQNTHVPRT (128) TNFNEKFKN (130) NTYLHWY (11) (129) (131)
Aho GSGYTFTNYY IGDINPVNGD TRGGYTMDY RSSQSLVHSNG LIYKVSYRFS SQNTHVPRT (15) TNFNEKFKN (16) NTYLHW (10) (20) (132) (19) Y AbM GYTFTNYYVF IGDINPVNGD TRGGYTMDY LIYKVSYRFS SQNTHVPRT (4) TNFNEKFKN (5) RSSQSLVHSNG (10) (11) (133 ) NTYLHWY (9) W Contact GYTFTNYYVF IGDINPVNGD TRGGYTMDY RSSQSLVHSNG LLIYKVSYRFS SQNTHVPRT (134) TNFNEKFKN (136) NTYLHWY (138) (139) (135) (137) (137)
Table 2. Amino acid sequence of anti-V domains
Murine CD47 VXP037 (mVH / mVL) and CDR grafts from human germline (VH1 / VL1). SEQ ID NOs are shown in parentheses.
1 Definitions of human germline used for grafting, based on the IMGT system. 2 CDR residues are in bold and underlined. As noted above, the “unified” CDR definitions used in this manuscript are an expanded definition compared to the classic Kabat definition.
Table 3. Amino acid sequences of unique CDRs (using unified definition) of 854 unique anti-CD47 v domains. SEQ ID NOs are shown in parentheses.
Lineage
Germinating DOMAIN Amino acid sequence2
Human V1
EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGDINPV CD47- n / a NGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQ mVH GTLVTVSS (140)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYYVFWVRQMPGKGLEWIGDINPV CD47- IGHV5-51 NGDTNFNESFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGTV VH1 (GT) 141
DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIY CD47- n / a KVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPRTFGQGTK 142 (VV)
DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIY CD47- IGKV2-28 KVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQGRK2R LCD1R1 RSSHSLVHSNG GYNFTNY MGDINPFNGDTNFNPSFQ KGSNRAS FQNTHTPRT GGFTMDY
NTYLH YIF G (67) (54) (66) (144) (145) (146) RSSQSFLHSNG GYRFTNY MGDINPGDGDTNFNPSFQ KGSNRFS LQNTHTPRT GGHTMDY
NNYLH YIF G (47) (148) (151) (147) (149) (150) RSSQSLLHSNG GYRFTNY MGDINPGNGDTNFNPSFQ KGSNRSS MQALHTPWT GGITMDY
NNYLD YVF G (153) (154) (157) (152) (155) (156) RSSQSLLHSNG GYSFNNY MGDINPGNGDTNYNPSFQ KGSYRAS MQALHVPRT GGQTMDQ
NNYLH YIF G (58) (159) (162) (158) (160) (161) RSSQSLLHSNG GYSFTNY MGDINPGNGDTNYSPSFQ KGSYRFS MQALQVPWT GGYIMDY
NTYLD YIF G (63) (164) (166) (163) (43) (165) RSSQSLLHSNG GYSFTSY MGDINPGNGDTRFNPSFQ KGSYRLS MQATHVPWT GGYTADY
NTYLH YIF G (168) (169) (172) (167) (170) (171) RSSQSLLHSNG GYSFTSY MGDINPGNGDTRFSPSFQ KVSNRAS MQATQVPWT GGYTLDY
SNYLH YIV G (174) (175) (178) (173) (176) (177) RSSQSLLHSNG GYSFTSY MGDINPGNGDTRYNPSFQ KVSNRFS MQNLQTPRT GGYTMDA
YNYLH YVF G (85) (179) (182) (46) (180) (181) RSSQSLLHSNG GYSFTSY MGDINPGNGDTRYSPSFQ KVSNRLS MQNTHIPRT GGYTMDE
YTYLD YVG G (53) (184) (187) (183) (185) (186) RSSQSLLHSNG GYTFTNY MGDINPGNSDTNFNPSFQ KVSYRAS MQNTHTLRT GGYTMDF
YTYLH YIF G (188) (189) (191) (52) (49) (190) RSSQSLVHSNG GYTFTSY MGDINPGNSDTNYNPSFQ KVSYRLS MQNTHTPRT GGYTMDH
NNYLD YIF G (193) (194) (197) (192) (195) (196) RSSQSLVHSNG GYTFTSY MGDINPVDGDTKYNPSFQ LGSNRAS MQNTHVPRT GGYTMDI
NNYLH YVF G (72) (198) (70) (201) (199) (200) RSSQSLVHSNG MGDINPVDGDTNFNPSFQ LGSNRFS MQNTQTPRT GGYTMDK
NTYLD G (71) (202) (56) (62) (203) RSSQSLVHSNG MGDINPVDGDTNFSPSFQ LGSNRLS MQTTHTPRT GGYTMDL
YNYLH G (77) (204) (206) (207) (205) RSSQSLVHSNG MGDINPVDGDTNYNPSFQ LGSYRAS MQTTQIPRT GGYTMDM
YTYLD G (208) (209) (211) (212) (210) RSSQSLVHSNG MGDINPVDGDTNYSPSFQ LGSYRFS SQATHFPRT GGYTMDN
YTYLH G (213) (214) (216) (57) (215)
LGSYRLS SQATQTPRT MGDINPVDGDTRFNPSFQ GGYTMDQ (217) (73) G (218) (219) MGDINPVDGDTRYNPSFQ LVSNRAS SQNIQTPRT GGYTMDR
G (220) (221) (51) (222) MGDINPVDGDTRYSPSFQ LVSNRFS SQNLHTPRT GGYTMDT
G (223) (224) (226) (225)
LVSNRLS SQNLQTPRT MGDINPVDSDTKFNPSFQ GGYTMDV (227) (228) G (229) (230)
LVSYRAS SQNMHTPRT MGDINPVDSDTNFNPSFQ GGYTMDW (231) (232) G (233) (234)
MGSNRFS SQNTHFPRT MGDINPVDSDTNYNPSFQ GGYTMGK (235) (236) G (237) (61)
MGSYRLS SQNTHVPWT MGDINPVDSDTNYSPSFQ GGYTPDY (238) (239) G (240) (45)
MVSNRFS SQNTQAPRT MGDINPVDSDTRFNPSFQ GGYTRDY (241) (242) G (243) (244)
SQNTQTPRT MGDINPVDSDTRYNPSFQ GGYTTDS (59) G (245) (246)
SQNTQTPWT MGDINPVDSDTRYSPSFQ GGYTTDW (247) G (248) (249)
SQNTQVPRT MGDINPVNGDTKYNPSFQ GGYTTDY (250) G (251) (252)
SQSTHVPRT MGDINPVNGDTNFSPSFQ GGYVMDY (253) G (254) (255)
SQTTHIPRT MGDINPVNGDTNYNPSFQ (256) G (257)
SQTTHVPRT MGDINPVNGDTNYSPSFQ (258) G (44)
SQTTQTPRT MGDINPVNGDTRFNPSFQ (259) G (260)
TQNTHTPRT MGDINPVNGDTRFSPSFQ (261) G (262)
VQNTQVPRT MGDINPVNGDTRYNPSFQ (263) G (264)
MGDINPVNGDTRYSPSFQ G (265)
MGDINPVNSDTKYNPSFQ G (266)
MGDINPVNSDTNFNPSFQ G (267)
MGDINPVNSDTNYSPSFQ G (268)
MGDINPVNSDTRFNPSFQ G (269)
MGDINPVNSDTRFSPSFQ G (270)
MGDINPVNSDTRYNPSFQ G (271)
MGDINPVNSDTRYSPSFQ G (272)
MGDIYPGNSDTKYNPSFQ G (273)
MGDIYPVNGDTRYNPSFQ G (274)
MGIINPGNGDTRYNPSFQ G (275)
MGIINPVDGDTKYNPSFQ G (276)
MGIINPVDGDTNYSPSFQ G (277)
MGIINPVDGDTRFNPSFQ G (278)
MGIINPVDGDTRFSPSFQ G (279)
MGIINPVDGDTRYNPSFQ G (74)
MGIINPVDGDTRYSPSFQ G (65)
MGIINPVDSDTNYNPSFQ G (280)
MGIINPVDSDTRFNPSFQ G (281)
MGIINPVDSDTRYNPSFQ G (282)
MGIINPVDSDTRYSPSFQ G (283)
MGIINPVNGDTKFNPSFQ G (284)
MGIINPVNGDTKYNPSFQ G (285)
MGIINPVNGDTKYSPSFQ G (286)
MGIINPVNGDTNFNPSFQ G (287)
MGIINPVNGDTNFSPSFQ G (288)
MGIINPVNGDTNYNPSFQ G (60)
MGIINPVNGDTNYSPSFQ G (289)
MGIINPVNGDTRFNPSFQ G (290)
MGIINPVNGDTRFSPSFQ G (291)
MGIINPVNGDTRYNPSFQ G (292)
MGIINPVNGDTRYSPSFQ G (293)
MGIINPVNSDTKYNPSFQ G (294)
MGIINPVNSDTNFNPSFQ G (295)
MGIINPVNSDTNYSPSFQ G (296)
MGIINPVNSDTRFSPSFQ G (297)
MGIINPVNSDTRYNPSFQ G (298)
MGIINPVNSDTRYSPSFQ G (299)
MGNINPVDGDTRYNPSFQ G (300)
MGVINPVNSDTNYNPSFQ G (301) Table 4. Amino acid sequences of CDRs (using unified definition) of unique anti-CD47 blocking CD47-SIRPα interaction IgGs, derived from libraries and designer. SEQ ID NOs are shown in parentheses.
LCDR1 name LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 clone SQNLHV GYSFTNY MGDINPVNGDTN
RSSQSLLHSNGYN KGSNRFS GGYTPDY D-H3 PRT YIF YSPSFQG YLH (46) (47) (45) (48) (43) (44) RSSQSLLHSNGYT FQNTHT GYTFTNY MGIINPVDGDTN
KVSNRLS GGYTMDR A-D5 YLH PRT YIF YNPSFQG (53) (51) (52) (54) (49) (50) RSSQSLVHSNGYT SQNTQT GYSFTNY IGDINPVNGDTN
KGSYRAS GGYTMDK G-B6 YLH PRT YIF FSPSFQG (58) (56) (57) (59) (43) (55) RSSQSLVHSNGNT SQATHT GYSFTNY MGIINPVNGDTN
KGSYRFS GGYTMGK F-E7 YLD PRT YIF YNPSFQG (63) (61) (62) (64) (43) (60)
RSSQSLLHSNGYN SQNTHT GYSFTNY MGIINPVDGDTR VH-A1 / VL- KGSNRAS GGFTMDY
YLH PRT YIF YSPSFQG B1 (67) (66) (46) (68) (43) (65) RSSQSLLHSNGYN SQNTQT GYSFTNY IGIINPVDGDTR LGSNRFS GGYTMDI
MH YLH PRT YIF YSPSFQG (71) (70) (46) (59) (43) (69) RSSQSLLHSNGYN SQATQT GYSFTNY MGIINPVDGDTR LGSNRAS GGYTMDI
TTP YLH PRT YIF YSPSFQG (72) (70) (46) (73) (43) (65) RSSQSLLHSNGYT FQNTHT GYSFTNY MGIINPVDGDTN
KVSNRLS GGYTMDR A-D5.1 YLH PRT YIF YNPSFQG (53) (51) (52) (54) (43) (50) RSSQSLLHSNGYT FQNTHT GYSFTNY MGIINPVDGDTR
KVSNRLS GGYTMDR A-D5.2 YLH PRT YIF YNPSFQG (53) (51) (52) (54) (43) (74) RSSQSLLHSNGYT FQNTHT GYSFTNY MGIINPVDGDTR
KVSNRLS GGYTMDR A-D5.3 YLH PRT YIF YSPSFQG (53) (51) (52) (54) (43) (65)
RSSQSLLHSNGYN KVSNRLS FQNTHT GYTFTNY MGIINPVDGDTN GGYTMDR A-D5.4 YLH (53) PRT YIF YNPSFQG (51)
(46) (54) (49) (50) RSSQSLLHSNGYN FQNTHT GYTFTNY MGIINPVDGDTN
KGSNRLS GGYTMDR A-D5.5 YLH PRT YIF YNPSFQG (75) (51) (46) (54) (49) (50) RSSQSLLHSNGYN FQNTQT GYTFTNY MGIINPVDGDTN
KGSNRLS GGYTMDR A-D5.6 YLH PRT YIF YNPSFQG (75) (51) (46) (76) (49) (50) RSSQSLLHSNGYN FQNTQT GYTFTNY MGIINPVDGDTN
LGSNRLS GGYTMDR A-D5.7 YLH PRT YIF YNPSFQG (77) (51) (46) (76) (49) (50) RSSQSLLHSQGYT FQNTHT GYTFTNY MGIINPVDGDTN
KVSNRLS GGYTMDR A-D5.8 YLH PRT YIF YNPSFQG (53) (51) (78) (54) (49) (50) RSSQSLLHSNGYT FQQTHT GYTFTNY MGIINPVDGDTN
KVSNRLS GGYTMDR A-D5.9 YLH PRT YIF YNPSFQG (53) (51) (52) (79) (49) (50) RSSQSLLHSQGYT FQQTHT GYTFTNY MGIINPVDGDTN
KVSNRLS GGYTMDR A-D5.10 YLH PRT YIF YNPSFQG (53) (51) (78) (79) (49) (50) Table 5. CDR amino acid sequences (using unified definition) of designer anti-CD47 IgGs, CD47-SIRPα interaction blockers, derived from A-D5. SEQ ID NOs are shown in parentheses.
Clone name LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 A-D5.11 RSSQSLLHSNGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTNYNP GGYTM
NYLH LS RT YIF SFQG DR (46) (53) (54) (43) (50) (51) A-D5.12 RSSQSLLHSNGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYNP GGYTM
NYLH LS RT YIF SFQG DR (46) (53) (54) (43) (74) (51) A-D5.13 RSSQSLLHSNGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (46) (53) (54) (43) (65) (51) A-D5.14 RSSQSLLHSSGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (80) (53) (54) (43) (65) (51) A-D5.15 RSSQSLLHSGGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (81) (53) (54) (43) (65) (51) A-D5.16 RSSQSLLHSAGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (82) (53) (54) (43) (65) (51) A-D5.17 RSSQSLLHSTGY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (83) (53) (54) (43) (65) (51) A-D5.18 RSSQSLLHSNAY KVSNR FQNTHTP GYSFTNY MGIINPVDGDTRYSP GGYTM
NYLH LS RT YIF SFQG DR (84) (53) (54) (43) (65) (51)

权利要求:
Claims (1)
[1]
1. Antibody molecule that specifically binds to human CD47 and CD47 of the cynomolgus monkey, and optionally also binds to mouse CD47, or a fraction of antigen binding thereof, characterized by the fact that the antibody molecule or the antigen-binding fraction comprises a variable region of the heavy chain with: an HCDR1 having amino acids in sequence in the following order: GSGYT / SFTNYY (SEQ ID NO: 30); an HCDR2 having amino acids in sequence in the following order: I-N-P-V-N / D-G-D-T-N / R-F / Y-N / S-P-S-F-Q-G (SEQ ID NO: 31); and an HCDR3 having amino acids in sequence in the following order: G-G-F / Y-T-M / P-D (SEQ ID NO: 32).
2. Antibody molecule or antigen-binding fraction, according to claim 1, characterized by the fact that it comprises a variable region of the light chain with: an LCDR1 having amino acids in sequence in the following order: SSQSLL / VHSN / Q / AGY / NN / TY (SEQ ID NO: 41); an LCDR2 having amino acids in sequence in the following order: L / K-V / G-S-N / Y-R-A / F / L-S (SEQ ID NO: 39); and an LCDR3 having amino acids in sequence in the following order: Q / N / A-T / L-Q / H-T / V-P-R (SEQ ID NO: 42).
3. Antibody molecule or antigen-binding fraction, according to claim 2, characterized by the fact that it comprises:
(a) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVNGDTNYSPSFQG (SEQ ID NO: 87) (HCDR2), GGYTPD
(SEQ ID NO: 88) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), KGSNRFS (SEQ ID NO: 47) (LCDR2) and NLHVPR (SEQ ID
NO: 90) (LCDR3) [Clone D-H3]; or
(b) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5]; or
(c) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVNGDTNFSPSFQG (SEQ ID NO: 94) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLVHSNGYTY (SEQ ID NO: 95)
(LCDR1), KGSYRAS (SEQ ID NO: 58) (LCDR2) and NTQTPR (SEQ ID
NO: 96) (LCDR3) [Clone G-B6]; or
(d) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVNGDTNYNPSFQG (SEQ ID NO: 97) (HCDR2), GGYTMG
(SEQ ID NO: 98) (HCDR3), SSQSLVHSNGNTY (SEQ ID NO: 19)
(LCDR1), KGSYRFS (SEQ ID NO: 63) (LCDR2) and ATHTPR (SEQ ID
NO: 99) (LCDR3) [Clone F-E7]; or
(e) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGFTMD (SEQ ID NO: 101) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO:
89) (LCDR1), KGSNRAS (SEQ ID NO: 67) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone VH-A1 / VL-B1]; or
(f) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO:
89) (LCDR1), LGSNRFS (SEQ ID NO: 71) (LCDR2) and NTQTPR (SEQ
ID NO: 96) (LCDR3) [MH Clone]; or
(g) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO:
89) (LCDR1), LGSNRAS (SEQ ID NO: 72) (LCDR2) and ATQTPR (SEQ
ID NO: 102) (LCDR3) [TTP Clone]; or
(h) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5.1]; or
(i) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO:
92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.2]; or
(j) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO:
92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.3]; or
(k) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5.4]; or
(l) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5.5]; or
(m) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), KGSNRLS (SEQ ID NO: 75) (LCDR2) and NTQTPR (SEQ ID
NO: 96) (LCDR3) [Clone A-D5.6]; or
(n) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), LGSNRLS (SEQ ID NO: 77) (LCDR2) and NTQTPR (SEQ ID
NO: 96) (LCDR3) [Clone A-D5.7]; or
(o) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5.8]; or
(p) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and QTHTPR (SEQ ID
NO: 105) (LCDR3) [Clone A-D5.9]; or
(q) the GSGYTFTNYY amino acid sequences (SEQ ID NO:
15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and QTHTPR (SEQ ID
NO: 105) (LCDR3) [Clone A-D5.10]; or
(r) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD
(SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89)
(LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID
NO: 93) (LCDR3) [Clone A-D5.11]; or
(s) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO:
89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.12]; or
(t) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO:
89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.13]; or
(u) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSSGYNY (SEQ ID NO:
106) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.14]; or
(v) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSGGYNY (SEQ ID NO:
107) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.15]; or
(w) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO:
108) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.16]; or
(x) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSTGYNY (SEQ ID NO:
109) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ
ID NO: 93) (LCDR3) [Clone A-D5.17]; or
(y) the GSGYSFTNYY amino acid sequences (SEQ ID NO:
86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2),
GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNAYNY (SEQ ID NO: 110) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A -D5.18]; or (z) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.16-DI]; or (z.1) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ NO: 92) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2) and NTHTPR (SEQ ID NO: 93) LCDR3) [Clone A-D5-DI].
4. Antibody molecule or antigen-binding fraction according to claim 1, characterized by the fact that it comprises: an HCDR1 having the amino acid sequence GSGYTFTNYY (SEQ ID NO: 15) or GSGYSFTNYY (SEQ ID NO: 86) ; an HCDR2 having the amino acid sequence INPVDGDTNYNPSFQG (SEQ ID NO: 91) or INPVDGDTRYSPSFQG (SEQ ID NO: 100); and an HCDR3 having the amino acid sequence GGYTMD (SEQ ID NO: 16) and, optionally, further comprising:
an LCDR1 having the amino acid sequence SSQSLLHSNGYNY (SEQ ID NO: 89) or SSQSLLHSNGYTY (SEQ ID NO: 92) or SSQSLLHSAGYNY (SEQ ID NO: 108); an LCDR2 having the amino acid sequence KVSNRLS (SEQ ID NO: 53) or KVSNRFS (SEQ ID NO: 85); and an LCDR3 with the amino acid sequence NTHTPR (SEQ ID NO: 93).
5. Antibody molecule or antigen binding fraction, according to claim 3 or claim 4, characterized by the fact that it comprises: (a) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG ( SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5]; or (b) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 16) 89) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.4]; or (c) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1), KVSNRLS (SEQ ID NO: 53) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.16]; or
(d) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1), INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108 ) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.16-DI]; or (e) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1), INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16) (HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 16 92) (LCDR1), KVSNRFS (SEQ ID NO: 85) (LCDR2) and NTHTPR (SEQ ID NO: 93) LCDR3) [Clone A-D5-DI].
6. Antibody molecule or antigen-binding fraction according to any of the preceding claims 1 to 5, characterized by the fact that it comprises one or more substitutions, deletions or insertions that remove a post-translational modification site, for example example, a glycosylation site, a deamination site, a phosphorylation site or an isomerization / fragmentation site.
7. Antibody molecule or antigen-binding fraction according to any one of the preceding claims 1 to 6, characterized by the fact that it is human, humanized or chimeric.
8. Antibody molecule or antigen binding fraction according to any one of the preceding claims 1 to 7, characterized by the fact that it comprises one or more human variable domain structural skeletons in which the CDRs have been inserted.
9. Antibody molecule or antigen-binding fraction according to any one of the preceding claims 1 to 8, characterized by the fact that it comprises an IGHV5-51 human germline structure in which the corresponding HCDR sequences have been inserted.
10. Antibody molecule or antigen-binding fraction according to any of claims 2 to 9, characterized in that it comprises a human germline structure IGKV2-28 in which the corresponding LCDR sequences have been inserted.
11. Antibody molecule or antigen-binding fraction according to any one of the preceding claims 1 to 10, characterized in that it comprises an immunologically inert constant region.
12. Antibody molecule or antigen-binding fraction according to any one of the preceding claims 1 to 11, characterized in that it is a Fab fragment, an F (ab) 2 fragment, an Fv fragment, a tetrameric antibody , a tetravalent antibody, a multispecific antibody (for example, a bivalent antibody), a single domain antibody (for example, VHH or VNAR, or a fragment of any of the same), a monoclonal antibody or a fusion protein.
13. Immunoconjugate characterized by the fact that it comprises the antibody molecule or an antigen-binding fraction thereof, as defined in any one of claims 1 to 12, linked to a therapeutic agent.
14. Nucleic acid molecule characterized by the fact that it encodes the antibody molecule or antigen-binding fraction thereof, as defined in any of claims 1 to 12.
15. Vector characterized by the fact that it comprises the nucleic acid molecule as defined in claim 14.
16. Host cell characterized by the fact that it comprises the nucleic acid molecule as defined in claim 14 or the vector as defined in claim 15.
17. Method for producing an anti-CD47 antibody and / or an antigen binding fraction characterized by the fact that it comprises the culture of the host cell as defined in claim 16 under conditions that result in the expression and / or production of the antibody and / or antigen-binding fraction and isolation of the antibody and / or antigen-binding fraction from the host cell or culture.
18. Pharmaceutical composition characterized by the fact that it comprises the antibody molecule or antigen-binding fraction thereof, as defined in any one of claims 1 to 12, or the immunoconjugate as defined in claim 13, or the nucleic acid molecule as defined in claim 14, or the vector as defined in claim 15.
19. Method for improving an immune response in an individual characterized by the fact that it comprises the administration of an effective amount of the antibody molecule or an antigen-binding fraction thereof, as defined in any of claims 1 to 12, or the immunoconjugate as defined in claim 13, or the nucleic acid molecule as defined in claim 14, or the vector as defined in claim 15, or the pharmaceutical composition as defined in claim 18.
20. Method for treating or preventing cancer in an individual characterized by the fact that it comprises the administration of an effective amount of the antibody molecule or an antigen-binding fraction thereof, as defined in any of claims 1 to 12, or the immunoconjugate as defined in claim 13, or the nucleic acid molecule as defined in claim 14, or the vector as defined in claim 15, or the pharmaceutical composition as defined in claim 18.
21. Method according to claim 20, characterized by the fact that cancer is selected from the group consisting of: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma and hematological tissue cancer.
22. Antibody molecule or antigen-binding fraction thereof, as defined in any of claims 1 to 12, or immunoconjugate, as defined in claim 13, or nucleic acid molecule, as defined in claim 14, or vector as defined in claim 15, or pharmaceutical composition as defined in claim 18, characterized by the fact that it is for use in the treatment of cancer.
23. The antibody molecule, or an antigen-binding fraction thereof, or the immunoconjugate, or the nucleic acid molecule, or the vector for use according to claim 22, or the treatment method according to claims 20 or 21, characterized by the fact that the antibody, antigen-binding fraction, immunoconjugate, nucleic acid, vector or pharmaceutical composition is for separate, sequential or simultaneous use in a combination combined with a second therapeutic agent, for example, an agent anticancer.
24. Use of an antibody molecule or antigen-binding fraction thereof, as defined in any one of claims 1 to 12, or an immunoconjugate, as defined in claim 13, or a nucleic acid molecule, as defined in claim 14, or a vector, as defined in claim 15, or a pharmaceutical composition as defined in claim 18, characterized by the fact that it is for the manufacture of a medicament for the treatment of cancer.
25. Method for treating or preventing an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease in an individual characterized by the fact that it comprises administering an effective amount of the antibody molecule or its antigen-binding fraction, as defined in any one of claims 1 to 12, or immunoconjugate as defined in claim 13, or the nucleic acid molecule as defined in claim 14, or the vector as defined in claim 15, or the pharmaceutical composition as defined in claim 18.
26. Method according to claim 25, characterized by the fact that autoimmune disease or inflammatory disease is selected from the group consisting of: arthritis, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave's disease and Hashimoto's thyroiditis and ankylosing spondylitis.
27. Antibody molecule or antigen-binding fraction thereof, as defined in any of claims 1 to 12, or immunoconjugate as defined in claim 13, or nucleic acid molecule, as defined in claim 14, or vector, as defined in claim 15, or pharmaceutical composition as defined in claim 18, characterized by the fact that it is for use in the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
28. Use of an antibody molecule or antigen-binding fraction thereof, as defined in any one of claims 1 to 12, or an immunoconjugate, as defined in claim 13, or a nucleic acid molecule, as defined in claim 14, or a vector,
as defined in claim 15, or a pharmaceutical composition as defined in claim 18, characterized by the fact that it is for the manufacture of a medicament for the treatment of an ischemia-reperfusion injury, an autoimmune disease or an inflammatory disease.
29. Method for treating or preventing cardiovascular disease (such as coronary heart disease or atherosclerosis) or fibrotic disease in an individual, characterized by the fact that it comprises the administration of an effective amount of the antibody molecule or a fraction of binding to the antigen thereof, as defined in any one of claims 1 to 12, or the immunoconjugate, as defined in claim 13, or the nucleic acid molecule, as defined in claim 14, or the vector, as defined in claim 15, or pharmaceutical composition as defined in claim 18.
30. Method according to claim 29, characterized by the fact that fibrotic disease is selected from the group consisting of myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis and asthma.
31. Antibody molecule or antigen-binding fraction thereof, as defined in any one of claims 1 to 12, or immunoconjugate, as defined in claim 13, or nucleic acid molecule, as defined in claim 14, or vector, as defined in claim 15, or pharmaceutical composition as defined in claim 18, characterized by the fact that it is for use in the treatment of cardiovascular disease (such as coronary heart disease or atherosclerosis) or fibrotic disease.
32. Use of an antibody molecule or antigen-binding fraction thereof, as defined in any of claims 1 to 12, or an immunoconjugate, as defined in claim 13, or a nucleic acid molecule, as defined in claim 14, or a vector, as defined in claim 15, or a pharmaceutical composition as defined in claim 18, characterized in that it is for the manufacture of a medicament for the treatment of cardiovascular disease (such as coronary heart disease or atherosclerosis) or a fibrotic disease.
33. Method for producing an antibody molecule that specifically binds to human CD47 and CD47 of cynomolgus monkeys and, optionally, also to mouse CD47, or to a fraction of binding to the antigen thereof, characterized by the fact that it comprises the steps of: (1) grafting anti-CD47 CDRs from a non-human source into a human v domain structure to produce a humanized anti-CD47 antibody molecule or antigen binding fraction thereof; (2) generating a phage library of clones of the humanized anti-CD47 antibody molecule or an antigen-binding fraction thereof, comprising one or more mutations in the CDRs; (3) screening the phage library for binding to human CD47 and monkey cynomolgus CD47, and optionally also to mouse CD47; (4) selecting clones from the screening step (3) having specificity of binding to human CD47 and CD47 of cynomolgus monkeys, and optionally also to mouse CD47; and (5) to produce an antibody molecule that specifically binds to human CD47 and cynomolgus monkey CD47, and optionally also to mouse CD47, or an antigen-binding fraction of the clones selected in step (4).
34. Method according to claim 33, characterized by the fact that it comprises an additional step of producing additional clones based on the clones selected in step (4), for example, based on additional exploratory mutagenesis at specific positions in the CDRs of the clones selected in step (4), to improve humanization and / or minimize the content of the human T cell epitope and / or improve the manufacturing properties in the antibody molecule or the antigen binding fraction produced in step ( 5).

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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