anti-cd47 antibodies and their uses

专利摘要:
the present invention relates to an antibody against cd47 and antibody fragments thereof, and to a composition containing the antibody or antibody fragments thereof. the present invention further relates to a nucleic acid encoding antibodies or antibody fragments thereof and host cells comprising them, as well as their relevant use. moreover, the present invention also relates to the use of these antibodies and antibody fragments in therapy and diagnosis.
公开号:BR112019014694A2
申请号:R112019014694
申请日:2018-08-28
公开日:2020-04-07
发明作者:Tsun Andy；Chen Bingliang；Liu Dandan；Liu Junjian
申请人:Innovent Biologics Suzhou Co Ltd；
IPC主号:

专利说明:

Patent Description for ANTI-CD47 ANTIBODIES AND USES OF THE SAME [0001] The present invention relates to a new antibody that specifically binds to the integrin-associated protein (IAP) (also called CD47) and its fragments of antibodies, and a composition comprising the antibody or antibody fragments. The present invention further relates to a nucleic acid encoding antibodies or their antibody fragments and host cells comprising them, as well as their relevant use. In addition, the present invention also relates to the use of these antibodies and antibody fragments in therapy and diagnosis.
BACKGROUND OF THE INVENTION [0002] Immunotherapy for cancer has been a highlight in the field of biological sciences in recent years and both therapies with T cell-based immunological control point inhibitors with CTLA4 antibodies, PD-L1 antibodies, etc. and cell therapies, such as CAR-T, TCR-T, etc. are very popular immunotherapies in recent years. These immunotherapies, without exception, focus on how to restore the functionality of T cells, in other words, focus mainly on how to improve the competence of the acquired immunity system. However, it is still tortuous for the path to beat cancers by targeting immunological control points and activating the functionality of T cells, thus increasing the competence of the acquired immunity system. However, the role of the intrinsic immune system in tumor immunotherapy has not been considered for a long time. In fact, macrophages represent about 50% of tumor tissue throughout the region of tumor infiltration. It is more important that the number of macrophages be inversely correlated with the prognosis of a tumor, which demonstrates the extremely important role of macrophages in tumors.
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2/99 [0003] Two signals are necessary for the phagocytic effect of macrophages: One signal is the activation of the eat me signal that targets the cell surface, another is the deactivation of the don’t eat me signal that targets the same surface. The absence of any of the signs is insufficient to trigger the onset of the phagocytic effect. The growing evidence shows that CD47 belongs to a class of don’t eat me signals and inhibits phagocytosis by macrophages interacting with the signal regulatory protein a (SIRP a) on the surface of the macrophage. Tumor cells can also prevent macrophage phagocytosis by expression of CD47 (see, for example, EP2242512 and the relevant literature mentioned there).
[0004] CD47 is also called an integrin-associated protein (IAP) and is a member of the immunoglobulin superfamily. CD47 is widely expressed on the surface of cells and can interact with SIRPa, thrombospondin-1 (TSP1) and integrins, mediating a combination of responses, such as apoptosis, proliferation, immunity, etc. TSP1 is related to cell proliferation, growth and differentiation. The binding of CD47 to TSP1 plays an important role in regulating cell migration, cell proliferation and apoptosis, and facilitates angiogenesis and the inflammatory response. In addition, CD47 is an important marker for self-recognition on the cell surface. CD47 can bind to the SIRPa protein on the surface of the macrophage, phosphorylate the protein tyrosine-based immunoreceptor (ITIM) inhibition motive, and subsequently recruit the SHP-1 protein, resulting in a series of cascade responses to inhibit macrophage phagocytosis (see, for example, Document US9382320 and relevant literature mentioned there).
[0005] Different studies show that almost all tumor tissues and cells express CD47 highly. CD47 highly expressed on the surface of don’t eat me signals from tumor cells
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3/99 by binding to SIRPa on the surface of macrophages, which allows macrophages in infiltrated areas of tumor tissue to not only be in harmony with tumor cells, but also to facilitate the proliferation of vessels within the tumor, inhibits the function of effector T cells, thus facilitating the proliferation and growth of tumor cells.
[0006] The role of CD47 in facilitating cell proliferation depends largely on cell types, as activation and loss of CD47 can result in greater proliferation. Activation of CD47 with TSP-1 may increase the proliferation of human U87 and U373 astrocytoma cells, but not normal astroglial cells. In addition, CD47 blocks the inhibitory effect of antibodies on the proliferation of unstimulated astrocytoma cells, but does not inhibit normal astroglial cells. Although the exact mechanism has not been elucidated, CD47 can facilitate the proliferation of cancer cells via the PI3K / Akt pathway, although it cannot facilitate the proliferation of normal cells (Sick E., Boukhari A., Deramaudt T., Rondé P ., Bucher B., André P., Gies JP, Takeda K., Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt ~ dependent pathway, Glia. February 2011; 59 (2): 308- 19: 308-19).
[0007] Binding to CD47 results in cell death of many normal and tumor cell lines through apoptosis or autophagy. Activation of CD47 induces rapid apoptosis of T cells. Incubation of Jurkat cells and peripheral blood mononucleated cells (PBMC) with monoclonal antibody Ad22 results in apoptosis within 3 hours. However, no cell apoptosis was observed after incubation with other anti-CD47 antibodies. The function of CD47 to induce apoptosis appears to be dependent on activation in specific epitopes in the extracellular domain (Rettersen R.D., Hestdal K., Olafsen M.
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K., Lie S. 0., Lindberg F. P. (June 1999), CD47 signals T cell death, J. Immunol., 162 (12): 7031-40. PMID 10358145).
[0008] Currently, a plurality of anti-CD47 antibodies have been reported. For example, a human chimeric monoclonal antibody of the lgG1 class derived from B6H12 and the humanized B6H12 antibody produced by CDR grafting were reported in US2015 / 0183874 A1, and had less immunogenicity compared to the known antibody. An anti-CD47 antibody that does not result in an apparent hemagglutination reaction has been reported in US Patent 9045541 and is significantly effective in a tumor model compared to the known antibody, for example, in increasing the ability of macrophages to phagocytize tumor cells.
[0009] Most of the antibodies known in the prior art that block the binding of CD47 to SIRPa cause agglutination of red blood cells, facilitating phagocytosis by macrophages, which significantly weakens the therapeutic effect of the corresponding antibody.
[0010] Thus, in the various therapies against tumors and / or cancers, there is a great need for the development of an anti-CD47 antibody that has good specificity for a target site, excellent therapeutic efficacy (for example, improvement of phagocytosis by macrophages, inhibition of tumor growth and even the possibility of complete disappearance of tumors) and fewer side effects. The present invention satisfies the need in this regard.
SUMMARY OF THE INVENTION [0011] The invention provides an anti-CD47 antibody, the composition, kit, method and use relevant to the anti-CD47 antibody.
[0012] The inventors of the invention made the surprising discovery that the antibody developed in the present invention has important anti-tumor activities, is capable of significantly inhibiting the
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5/99 tumor growth, and even allows the tumor to completely disappear.
[0013] In some embodiments, the invention provides an anti-CD47 antibody that binds to a CD47 or a fragment thereof (preferably a human CD47 protein) or its antibody fragment (preferably the antigen binding fragment thereof) ).
[0014] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises or consists of the sequence of one to three of the following heavy chain complementarity determining regions (HCDRs) selected from among the group consisting of: (i) HCDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 and 99, (ii) HCDR2 comprising the sequence of amino acids selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 and 101, (iii) HCDR3 comprising the sequence of amino acids selected from the group that consists of SEQ ID NO: 17, 18, 19, 20, 21, 22, 102 and 103, and (iv) HCDRs in (i), (ii) and (iii), comprising substitution of an amino acid (for example, conservative substitution), elimination or insertion of at least one amino acid and not more than 5 amino acids, in which the anti -CD47, comprising the modified CDRs still has the ability to bind to CD47.
[0015] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof in accordance with the present invention comprises or consists of the sequence of one to three of the following light chain complementarity determining regions (LCDRs) selected from among the group consisting of: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 23 and 24, (ii) LCDR2 comprising the amino acid sequence of SEQ ID:
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NO: 25 and 26, (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, 28, 29 and 30, and (iv) LCDRs in (i), (ii) and (iii), comprising the replacement of an amino acid (for example, a conservative substitution), deletion or insertion of at least one amino acid and not more than 5 amino acids, wherein the anti-CD47 antibody, comprising the modified CDRs, still has the ability to bind to CD47.
[0016] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises A) one to three of the following heavy chain complementarity determining regions (HCDRs) selected from the group that consists of: (i) HCDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 and 99; (li) HCDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 and 101; (iii) HCDR3 comprising the sequence of amino acids selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 102 and 103; and (iv) HCDRs in (i), (ii) and (iii), comprising the substitution of an amino acid (for example, a conservative substitution), deletion or insertion of at least one amino acid and not more than 5 amino acids; and B) one to three of the following light chain complementarity determining regions (LCDRs) selected from the group consisting of: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 23 and 24, (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 25 and 26, (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, 28, 29 and 30, and (iv) LCDRs in (I), (ii) and ( iii), comprising the substitution of an amino acid (for example, a conservative substitution), deletion or insertion of at least one amino acid and not more than 5 amino acids,
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7/99 wherein the anti-CD47 antibody, comprising the modified CDRs, still has the ability to bind CD47.
[0017] In some embodiments, the anti-CD47 antibody or antigen-binding fragment according to the present invention comprises the HCDR1, HCDR2 and HCDR2 heavy chain complementarity determining regions, where HCDR1 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 and 99; HCDR2 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 and 101; HCDR3 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21,22, 102 and 103.
[0018] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises the complementary determining regions of the light chain LCDR1, LCDR2 and LCDR3, where LCDR1 comprises or consists of the sequence amino acid selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 23 and 24; LCDR2 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 25 and 26; LCDR3 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 and 30.
[0019] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises the HCDR1, HCDR2 and HCDR3 heavy chain complementarity determining regions and the light chain complementarity determining regions , where HCDR1 with
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8/99 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 and 99; HCDR2 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 and 101; HCDR3 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 102 and 103; LCDR1 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 23 and 24; LCDR2 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 25 and 26; LCDR3 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 and 30.
[0020] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising the HCDR1, HCDR2 and HCDR3 heavy chain complementarity determining regions and the LCDR1 light chain complementarity determining regions, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 98 or 99; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 100 or 101; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 102 or 103; LCDR1 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 23 and 24; LCDR2 comprises or consists of the amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 25 and 26; LCDR3 comprises or consists of
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9/99 amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 and 30.
[0021] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27.
[0022] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27.
In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; HCDR3 with
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10/99 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27.
In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28.
[0025] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28.
[0026] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof,
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11/99 wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28.
[0027] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.
[0028] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; and LCDR3 comprises or consists of the amino acid sequence shown in
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SEQ ID NO: 29.
[0029] In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30.
In the preferred embodiment, the present invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30.
[0031] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof in accordance with the present invention comprises a variable region of the HCVR heavy chain that comprises or consists of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, 48, 49,
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50, 51, 52 and 53. In some embodiments, the HCVR heavy chain variable region of the anti-CD47 antibody comprises an amino acid sequence having one or more amino acid substitutions (for example, conservative substitutions), insertions or exclusions with respect to amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52 and 53, however, the anti-CD47 antibody comprising said HCVR still has the ability to bind to CD47.
[0032] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof in accordance with the present invention comprises a variable region of the HCVR light chain that comprises or consists of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 54, 55, 57 and 58. In some embodiments, the LCVR light chain variable region of the anti-CD47 antibody comprises an amino acid sequence having one or more amino acid substitutions (for example, conservative substitutions), insertions or deletions with respect to the amino acid sequence shown in SEQ ID NO: 54 , 55, 57 and 58, however, the anti-CD47 antibody comprising said LCVR still has the ability to bind to CD47.
[0033] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR heavy chain variable region comprises or consists of an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52 and 53 and the variable region of the light chain
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LCVR comprises or consists of an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 54, 55, 57 and 58.
[0034] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 44; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 54.
[0035] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 45; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 54.
[0036] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 46; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 54.
[0037] In the preferred embodiment, the invention provides an antLCD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 47; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 55.
[0038] In the preferred embodiment, the invention provides an anti-LCD47 antibody or antigen-binding fragment thereof, wherein the
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HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 48; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 55.
[0039] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 49; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 55.
[0040] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 50; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 57.
[0041] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 51; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 57.
[0042] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 52; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 58.
[0043] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the
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16/99 HCVR heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 53; and the LCVR light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 58.
[0044] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 74, 76 , 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97. In some embodiments, the heavy chain of the anti-CD47 antibody comprises an amino acid sequence having one or more amino acid substitutions (for example, conservative substitutions), insertions or deletions in relation to the amino acid sequence selected from the group consisting of SEQ ID NO: 74, 76, 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97, however, the antiCD47 antibody comprising said heavy chain still has the capacity age to bind to CD47.
[0045] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises a light chain, wherein the light chain comprises or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 75, 79, 83 and 86. In in some embodiments, the anti-CD47 antibody light chain comprises an amino acid sequence having one or more amino acid substitutions (for example, conservative substitutions), insertions or deletions with respect to the ami sequence
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17/99 noacids selected from the group consisting of SEQ ID NO: 75, 79, 83 and 86, however, the anti-CD47 antibody comprising said light chain still has the ability to Hgar to CD47.
[0046] In some embodiments, the anthCD47 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence with at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 74 , 76, 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97 and the light chain comprises or consists of a sequence of amino acids with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 75, 79 , 83 and 86.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 74; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
[0048] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 76; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
[0049] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 77; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
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In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 78; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 80; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
[0052] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 81; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
[0053] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 82; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 83.
[0054] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 84; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 83.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 86.
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In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 87; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 86.
[0057] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 88; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
[0058] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 89; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 90; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 75.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 91; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
[0061] In the preferred embodiment, the invention provides an antkCD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 92; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
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In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 93; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 79.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 94; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 83.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 95; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 83.
[0065] In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 96; and the light dog comprises or consists of the amino acid sequence shown in SEQ ID NO: 86.
In the preferred embodiment, the invention provides an anti-CD47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 97; and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 86.
[0067] In some embodiments, the antibody according to the invention also covers the amino acid sequence variants of the anti-CD47 antibody, the competing antibodies with any of the antibodies described above for binding to CD47, and the antibodies that bind to them epitopes of CD47, like any of the antibodies
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21/99 wells described above.
[0068] In some embodiments, the anti-CD47 antibody is a monoclonal antibody. In some embodiments, the anti-CD47 antibody is humanized. In some embodiments, the anti-CD47 antibody is a human antibody. In some embodiments, at least part of the anti-CD47 antibody structural sequence is a human consensus structural sequence. In one embodiment, the anti-CD47 antibody of the invention also comprises its antibody fragments, preferably selected from the group consisting of the following antibody fragments: Fab, Fab'-SH, Fv, scFv or (Fab ') 2 fragment .
[0069] In some embodiments, the anti-CD47 antibody of the invention is a blocking antibody that blocks the binding of CD47 to SIRPa.
[0070] In one aspect, the invention provides nucleic acids that encode any of the anti-CD47 antibodies or fragments thereof mentioned above. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of yeast cells, mammalian cells or other cells that are suitable for preparing the antibodies or their antigen-binding fragments. In another embodiment, the host cells were prokaryotic.
[0071] In one embodiment, the present invention provides a method of preparing an anti-CD47 antibody or fragment (preferably an antigen binding fragment), wherein the method comprises culturing the host cell under a condition that is suitable to express the nucleic acid encoding said anti
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22/99 body or its fragment (preferably, the antigen-binding fragment), and optionally isolating the antibody or its fragment (preferably, the antigen-binding fragment). In a given embodiment, the method further comprises recovering the anti-CD47 antibody or its fragment (preferably, the antigen-binding fragment) from the host cell.
[0072] In one embodiment, the present invention provides the anti-CD47 antibody or its fragment prepared by the method of the invention.
[0073] In some embodiments, the invention provides a composition comprising any of the anti-CD47 antibodies or fragments described herein (preferably, their antigen binding fragments), preferably the composition that serves as a pharmaceutical composition. In one embodiment, the composition also comprises pharmaceutical carriers.
[0074] In one aspect, the present invention is directed to a method of inhibiting the binding of CD47 to SIRPa in an individual, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments thereof. The present invention is also directed to the use of any of the antiCD47 antibodies or fragments described herein in the preparation of a composition or medication to inhibit the binding of CD47 to SIRPa in an individual.
[0075] In one aspect, the present invention is directed to a method of facilitating macrophage phagocytosis in an individual, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments thereof. The present invention is directed to the use of any of the anti-CD47 antibodies or fragments described herein in the preparation of a composition or medication to facilitate phagocytosis by macrophages in
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23/99 an individual. In one embodiment, the anti-CD47 antibody of the invention can increase phagocytosis by macrophages by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% compared to a control.
[0076] In the other aspect, the present invention is directed to a method of treating a CD47-related disorder in an individual, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments thereof. The present invention is directed to the use of any of the anti-CD47 antibodies or fragments thereof described herein in the preparation of a medication to treat a CD47-related disorder in an individual.
[0077] In some embodiments, CD47-related disorder is a variety of hematological disorders and solid tumors, including, but not limited to, acute myelocytic leukemia (AML), chronic myelocytic leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin's disease (NHL ), multiple myeloma (MM), lymphoma, breast carcinoma, gastric carcinoma, lung cancer, esophageal carcinoma, intestinal carcinoma, ovarian carcinoma, cervical carcinoma, renal carcinoma, pancreatic carcinoma, bladder carcinoma, glioma, melanoma and others solid tumors.
[0078] In one aspect, the present invention is directed to a method of immunotherapy in tumors with CD47 being the target, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments described herein. The present invention is further directed to the use of any of the anti-CD47 antibodies or fragments described herein in the preparation of a medication to treat a tumor.
[0079] In one aspect, the present invention is directed to a method of treating any disease or disorder that can be improved, delayed, inhibited or prevented by eliminating, re
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24/99 reduction or decrease in CD47 activity.
[0080] In another aspect, the method according to the present invention is further directed to a method of treating a tumor with a combination therapy, comprising administering to an individual the effective amount of any of the anti-CD47 antibodies or fragments thereof described here and one or more other medicines. In some embodiments, the method described herein further comprises co-administering to an individual an effective amount of the second drug, while the anti-CD47 antibody or its fragment described herein is the first drug. In one embodiment, the second drug is a chemotherapeutic agent, radiotherapeutic agent or biomacromolecular drug to treat the relevant diseases. In one embodiment, the biomacromolecular drug is, for example, several monoclonal antibody drugs that attack tumor cells through recognition by T cells, for example, rituximab, cetuximab and trastuzumab. The term second drug, as used in this document, cannot be interpreted as just a single type of drug other than the first drug. Thus, the second drug does not have to be a drug, but it may constitute or include more than one of these drugs.
[0081] In some embodiments, the individual or the individual is a mammal, preferably a human being.
[0082] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided for in the present invention may be effective in facilitating macrophage phagocytosis.
[0083] In a preferred embodiment, the anti-CD47 antibody or antigen-binding fragment thereof provided for in the present invention is surprisingly capable of effectively inhibiting tumor growth as compared to a control antibody.
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25/99 [0084] In a more preferred embodiment, the anti-CD47 antibody or antigen-binding fragment of the same provided for in the present invention allows for complete tumor regression, which is totally unexpected and has never been reported in the prior art.
[0085] In one aspect, the present invention is directed to a method of detecting the CD47 protein in a sample, comprising (a) bringing the sample into contact with any of the anti-CD47 antibodies or fragments described herein; and (b) detecting the formation of the complex between the anti-CD47 antibody or antigen-binding fragment thereof and the CD47 protein. In some embodiments, CD47 is a human CD47. In one embodiment, the detection method can be an in vitro or in vivo method. In one embodiment, ο anti-CD47 antibody is used to select individuals eligible for therapy with an anti-CD47 antibody. In one embodiment, the anti-CD47 antibody is detectably labeled.
[0086] In another aspect, the present invention is directed to a method for determining the effectiveness of a tumor therapy, comprising the step of determining the number of cancer cells that express CD47 in a sample from an individual before and after therapy, where the reduced number of cancer cells that express CD47 indicates that the therapy is effective.
[0087] The present invention also encompasses any combination of any of the modalities described herein. Any of the modalities or any combination of them described herein is applicable to each and all anti-CD47 antibodies or fragments, methods and uses of the invention described in this document.
BRIEF DESCRIPTION OF THE FIGURES [0088] Figure 1. Affinity assay with flow cytometry on an anti-CD47 antibody in IgG1 format produced in yeasts at the cellular level.
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26/99 [0089] Figure 2. Blocking the binding of SIRPa to a CD47 expressed in CHO cells by the present antibody in the lgG1 format produced in yeast cells, as tested with flow cytometry.
[0090] Figure 3. Blocking the binding of SIRPa to a CD47 expressed in CHO cells by the present antibody in the lgG4 format produced in CHO cells, as tested with flow cytometry.
[0091] Figure 4. Detection of the ability of the present antibody in the lgG1 format produced in yeast cells to facilitate phagocytosis of tumor cells by macrophages, the antibody including antibodies matured with antibody affinity to ADI-29336, ADI-29340, ADI- 29341 and ADI-29349 in lgG1 format produced in yeast cells.
[0092] Figure 5. Detection of the ability of the present IgG1 antibody produced in yeast cells to facilitate phagocytosis of tumor cells by macrophages.
[0093] Figure 6. Detection of the ability of the present antibody in the lgG4 format produced in CHO cells to facilitate phagocytosis of tumor cells by macrophages.
[0094] Figure 7. Detection of the ability of the present antibody in the lgG4 format produced in CHO cells to facilitate phagocytosis of tumor cells by macrophages. Included are ADI-29336, ADI29340, ADI-29341 and ADI-29349 and ADI-29371.
[0095] Figure 8. Study on the antitumor activity of the present antibody ADI-26630 in lgG4 format produced in CHO cells in a mouse model (tumor model NOD / SCID Raji). In which Figure 8B is an enlarged partial view of Figure 8A.
[0096] Figure 9: Study on the antitumor activity of the present antibody ADI-26624 in lgG4 format produced in CHO cells in a mouse model (tumor model NOD / SCID Raji).
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27/99 [0097] Figure 10: Study on the antitumor activity of the present antibodies ADI-26630, ADI29340 and ADI29341 in lgG4 format produced in CHO cells at a dosage of 0.5 mg / kg in a mouse model (tumor model NOD / SCID Raji).
[0098] Figure 11: Study on the antitumor activity of the present antibodies ADI-26630, ADI29340 and ADI29341 in lgG4 format produced in CHO cells at a dosage of 5 mg / kg in a mouse model (tumor model NOD / SCID Raji).
[0099] Figure 12: Result of the detection of the activity of an anti-CD47 antibody of the invention to facilitate the RBC agglutination activity.
DETAILED DESCRIPTION
1.1 DEFINITION [00100] Before the present invention is detailed below, it should be understood that the present invention is not limited to the particular methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used here is intended to describe the particular modalities only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by those skilled in the art to which the invention belongs.
[00101] For interpretation of the specification, the following definitions will be applied and, whenever necessary, the terms used in the singular can also include the plural, and vice versa. It should be understood that the terminology used here is intended to describe particular modalities only, and should not be considered limiting.
[00102] The term about, used in the combination of a naked value
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28/99 numeric, must cover numerical values in a range from a lower limit lower than the numerical value specified by 5% to an upper limit greater than the numerical value specified by 5%.
[00103] The term conservative substitution refers to the replacement of one amino acid by another amino acid in the same class, for example, the replacement of an acidic amino acid by another acidic amino acid, the replacement of a basic amino acid by another basic amino acid, and the replacement of one neutral amino acid for another neutral amino acid. Exemplary substitutions are shown in the Table below:
Original Residue Exemplary replacement Preferred replacement Wing (A) Go; Read; He Go Arg (R) Lys; Gin; Ãn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) To be; Allah To be Gin (Q) Ãsn; Glu Ãn Glu (E) Asp; Gin Asp Gly (G) Allah Allah His (H) Asn; Gin; Lys; Arg Arg He (1) Read; Go; Met; Allah; Phe; Norleucine Read Leu (L) Norleucine; lie; Go; Met; Allah; Phe He Lys (K) Arg; Gin; Asn Arg Met (M) Read; Phe; lie Read Phe (F) Trp; Read; Go; He; Allah; Tyr Tyr Pro (P) Oh Allah Being (S) Thr Thr Thr (T) Go; To be To be Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; To be Phe Go (V) He; Read; Met; Phe; Allah; Norleucine Read
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29/99 [00104] The term antibody is used here in the broadest sense and encompasses several antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity. An intact antibody will generally comprise at least two complete heavy chains and two complete light chains, although, in certain circumstances, it may comprise fewer chains, for example, naturally occurring antibodies in camels may comprise only heavy chains.
[00105] The term antigen-binding portion, as used in this document, refers to a portion that specifically binds to a target antigen. The term encompasses antibodies and other natural molecules (for example, receptors, ligands) or synthetic molecules (for example, DARPins) that specifically bind to a target antigen. In a preferred embodiment, the antigen-binding portion of an antibody according to the invention is an antibody fragment.
[00106] The terms full antibody, intact antibody and whole antibody are used interchangeably herein to refer to an antibody with a structure very similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
[00107] As used herein, the terms monoclonal antibody and monoclonal antibody composition refer to a preparation of antibody molecules of single amino acid composition, and should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies or antigen-binding fragments can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies, for example, see
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30/99 with CDR, or combinations of these or other technologies known in the art.
[00108] As used in this document, the terms bind to and specifically bind to refer to an antibody or antigen-binding portion that binds to an antigenic epitope in an in vitro assay, preferably in a light biointerferometry (ForteBio) using a purified wild-type antigen. In some embodiments, an antibody or antigen-binding portion is said to bind specifically to an antigen preferably when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules.
[00109] Depending on the amino acid sequence of the heavy chain constant region, the antibodies are divided into Class: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses, for example, lgG1, lgG2, lgG3 and lgG4, lgA1 as well as lgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called α, δ, ε, y μ. The light chain (CL) regions that can be found in all five classes of antibodies are called kappa and lambda. Within complete light and heavy chains, the variable and constant regions are usually joined by a J region of approximately 12 or more amino acids, and the heavy chain also includes a D region of about 10 or more amino acids. See, eg, Fundamental Immunology, Chapter 7 (Paul, W. Ed., 2nd edition, Raven Press, NY (1989)) (which is incorporated herein by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair normally form the antigen-binding sites.
[00110] The term variable region or variable domain refers to the domain of an antibody that is involved in binding the antibody
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31/99 to the antigen. The variable domains of the heavy chain and light chain of a native antibody generally have similar structures, with each domain comprising four conserved structural regions (FRs) and three complementarity determining regions (see, for example, Kindt et a., Kuby Immunology, 6th Edition, WH Freeman and Co., page 91 (2007)). A single VH and VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain of an antibody that binds to the antigen to screen a library of complementary VH or VL domains, respectively. See, for example, Portolano, et al., J. Immunol. 150. 880-887 (1993); Clarkson, et al., Nature 352: 624-628 (1991).
[00111] The variable regions usually have the same general structure as structural regions (FRs) relatively conserved joined by three hypervariable regions, the latter also called complementarity determining regions or CDRs. In general, the CDRs of the two chains of each pair are aligned with the structural regions, whose CDRs allow specific binding to an epitope. From the N ~ terminal to the C ~ terminal, two variable regions of the light and heavy chains normally comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains.
[00112] Complementary determining regions or CDR regions or CDRs or hypervariable regions (which can be used interchangeably with HVR hypervariable regions) is a region of amino acids in a variable region of antibodies, which is mainly responsible for binding to an epitope of a antigen. Heavy and light chain CDRs are usually called CD1, CD2 and CD3, numbered sequentially from the N-terminal. CDRs located in the variable domain of a year's heavy chain
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32/99 antibodies are referred to as HCDR1, HCDR2 and HCDR3, and CDRs located in the variable domain of an antibody light chain are referred to as LCDR1, LCDR2 and LCDR3.
[00113] Methods and techniques for identifying CDR sequences for a given VH or VL are well known in the art: Kabat Complementarity Determining Regions (CDRs) are identified based on sequence variability and are the most commonly used (Kabat et ak, Sequences of Proteins of Immunological Interest, 5th Edition Health Service Public, National Institutes of Health, Bethesda, Md. (1991), the definition of Chothia refers, conversely, the location of the structural loops (Chothia, et al (1987) J. Mol. Biol. 196: 901-917; Chothia, et al. (1989) Nature 342: 877-883), HVR AbM is a compromise between Kabat's HVRs and Chothia's structural loops, and are used by Oxford Molecular's AbM antibody modeling software, Contact HVR (Contact) is based on the analysis of a complicated crystalline structure available. According to the different conventions for identifying CDRs, each of the HVR / CDR residues in this s HVRs are described as follows.
CDR Definition ofKabat Definition ofAbM Definition ofChothia Definition ofContact LCDR1 L24-L34 L24-L34 L26-L32 L30-L36 LCDR2 L50-L56 L50-L56 L50-L52 L46-L55 LCDR3 L89-L97 L89-L97 L91-L96 L89-L96 HCDR1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering system) HCDR1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering system) HCDR2 H50-H65 H50-H58 H53-H55 H47-H58 HCDR3 H95-H102 H95-H102 H96-H101 H93-H101 (Kabat numbering system)
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33/99 [00114] In one embodiment, the CDR of an antibody of the invention has the CDR sequence located at the position of the Kabat residue below, according to the Kabat numbering system:
Positions 24-34 (LCDR1), Positions 50-56 (LCDR2) and Positions 8997 (LCDR3) in VL, and Positions 27-35 (HCDR1), Positions 50-65 (HCDR2), and Positions 93-102 ( HCDR3) in the VH.
[00115] A CDR can also be identified based on the position with the same Kabat number as a sequence of the reference CDR (for example, one of the exemplary CDRs of the invention).
[00116] An antibody fragment refers to a molecule other than an intact antibody, which comprises a part of an intact antibody that binds the antigen to which the intact antibody binds.
[00117] Affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise specified, when used in this document, binding affinity refers to the intrinsic binding affinity that reflects a 1: 1 interaction between the elements of a binding pair (for example, an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
[00118] The term compete when used in the context of antigen-binding proteins (eg neutralizing antigen-binding proteins and neutralizing antibodies) that compete for the same epitope means competition between antigen-binding proteins, which is determined by an assay below: the antigen-binding protein to be tested (for example, an antibody or immunologically functional antibody) in the assay prevents or inhibits (for example,
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34/99 reduces) the specific binding of a binding protein to the reference antigen (for example, a ligand or reference antibody) to a common antigen (for example, a CD47 or its fragment). A series of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. For example, these assays are direct or indirect solid phase radio-immunoassay (RIA), direct or indirect solid phase immunoassay (EIA), sandwich competition assay (for example, see Stahli et a!., 1983, Methods in Enzymology 9 : 242-253). Typically, the assay involves the use of a purified antigen bound to a solid surface or cells loaded with any of an unlabeled antigen binding protein to be tested and a tagged reference antigen binding protein. Concurrent inhibition is measured by determining the amount of the marker bound to the solid surface or cells in the presence of the antigen-binding protein to be tested. Generally, the antigen-binding protein to be tested is present in excess. The antigen-binding protein Identified by the competitor assay (the competing antigen-binding protein) includes: the antigen-binding protein that binds to the same epitope as the reference antigen-binding protein; and the antigen-binding protein that binds to an adjacent epitope sufficiently proximal to the epitope bound by the antigen-binding protein so that the mutual steric impediment of the two eplopes occurs. Additional details on the methods for determining the concurrent connection are provided in the Examples in this document. Generally, when present in excess, a competing antigen-binding protein will inhibit (for example, reduce) the specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45-50 %, 50-55%, 55-60%, 6065%, 65-70%, 70-75% or 75% or more. In one case, binding is inhibited
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35/99 is less 80-85%, 85-90%, 90-95%, 95-97% or 97% or more.
[00119] A human antibody is one that has an amino acid sequence that corresponds to an antibody generated by a human or a human cell or derived from a non-human source that uses human antibody repertoires or other human antibody coding sequences . This definition of a human antibody explicitly excludes a humanized antibody comprising non-human antigen-binding residues.
[00120] A human consensus framework refers to a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH structural sequences. Generally, the selection of human immunoglobulin VL or VH sequences is a selection from a subtype of variable domain sequences. Generally, the following subtype is one in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one modality, for VL, the subtype is the subtype layer I, such as Kabat and f /, (see above). In one modality, for VH, the subtype is the subtype cover III, as in Kabat ef a /, (see above).
[00121] A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will include substantially all of at least one, and usually two, variable domains, where all or substantially all of the HVRs (for example, CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody can optionally comprise at least a part of an antibody constant region derived from
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36/99 a human antibody, A humanized form of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.
[00122] The term diabody refers to antibody fragments with two antigen-binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VHVL ). With the use of a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain, in order to create two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in more detail, for example, in EP 404,097; WO 1993/01161; Hudson, et at., Nat. Med. 9: 129-134 (2003); and Hollinger, et al., Proc. Natl. Acad. Know. USA 90: 6444-6448 (1993). Tricorpos and Teracorpos are also described in Hudson, et al., Nat. Med. 9: 129134 (2003).
[00123] Effector Functions refer to biological activities that are attributable to the Fc region of an antibody and that vary according to the antibody's isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); binding of the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example, cell receptor
B); and activation of B cells.
[00124] The terms effective amount ”and therapeutically effective amount refer to an amount or dosage of the antibody or its antigen-binding fragment of the invention which, after administered to an individual in a single or several doses, generates the expected effects in the treated individual, including improved
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37/99 disorder of the individual (for example, improvement of one or more symptoms) and / or delay in the progression of symptoms and the like. Effective amount and therapeutically effective amount may also refer to an amount sufficient to decrease CD47 signals (for example, see Yamauchi, et a!., 2013 Blood, January 4; SotoPantoja, et al., 2013 Expert Opin Ther Targets , 17: 89-103; Irandoust, et al., 2013 PLoS One, Epub 8 de Janeiro; Chao, et al., 2012 Curr Opin Immunol, 24: 225-32; Theocharides, et al., 2012 J Exp Med, 209 (10): 1883-99), for example, an amount of an antibody sufficient to reduce the signal for inhibiting phagocytosis generated from the interaction between CD47 / SIRPa on the CD47 / SIRPa signal axis in macrophages, that is, the antibody of the invention facilitates macrophage-mediated phagocytosis of cells expressing CD47.
[00125] In one embodiment, the effective amount of the anti-CD47 antibody of the invention can improve / increase macrophage phagocytosis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 100% compared to a control.
[00126] Therapeutically effective amounts can be easily determined by the responsible physicians as well as those skilled in the art, considering a variety of the following factors: the species of the mammal; size, age and general health; the disease involved; the extent or severity of the disease; the response of an individual patient; the specific antibody administered; the mode of administration; the bioavailability profile of the formulation administered; the selected dose regimen; and the use of any concomitant therapy, etc.
[00127] As described above, in certain circumstances, the interaction between the antibody and its target antigen will interfere with the target's functionality. In addition, the dose of administration required is not dependent only on the binding affinity of an antibody
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38/99 to its specific antigen, but also to the clearance rate of an antibody given to an administered individual. As a non-limiting example, the therapeutically effective dose of an antibody or an antibody fragment of the invention is generally in the range of about 0.1 mg / kg of body weight to about 100 mg / kg of body weight. In some embodiments, the antibody of the invention is administered to an individual at a dose of 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg , 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg or more. The frequency of the common dose varies, for example, from twice a day to once a week, once every two weeks, once every three weeks, once a month, once every two months, once a week. every three months, once every six months.
[00128] The term block used here indicates the decrease in CD47 signaling in the presence of the antibody of the invention. Blocking of CD47-mediated signaling means that the level of CD47 signaling in the presence of the anti-CD47 antibody of the invention is lower than the signaling level of the control CD47 (namely, the level of CD47 signaling in the absence of the antibody of the invention) , with the decrease interval greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99 % or 100%. The level of CD47 signaling can be measured with many conventional techniques, such as reporter luciferase gene assays, which measure downstream gene activation and / or activation in response to CD47. One skilled in the art will understand that the signaling level of the CD47 can be measured with many tests, including, for example, commercially available kits.
[00129] The terms host cell, host cell line and host cell culture are used interchangeably and refer to a cell to which the exogenous nucleic acid was
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39/99 introduced, including the progeny of that cell. Host cells include transformants and transformed cells, which include the main transformed cell and its progeny, regardless of the number of passages. The offspring may not be entirely identical in nucleic acid content to a cell of origin, but may contain mutations. Mutant progeny that have the same biological function or activity as triad or selected in the originally transformed cell are included in this document.
[00130] The term cytotoxic agent is used here to refer to a substance that inhibits or prevents a cell function and / or causes cell death or destruction.
[00131] The term vector, when used in this document, denotes a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell to which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. These vectors are referred to here as expression vectors.
[00132] An immunoconjugate is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.
[00133] An individual (individual or subject) includes a mammal. Mammals include, but are not limited to, domestic animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates, such as monkeys), rabbits and rodents (for example, mice and rats). In some modalities, the individual is a human being.
[00134] An isolated antibody is one that has been separated from a component of its natural environment. In some modalities, a
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40/99 antibody is purified to purity greater than 95% or 99%, as determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, phase HPLC reverse or ion exchange). For review of methods for assessing antibody purity, see, for example, Flatman etaL, J. Chromatogr. B848: 79-87 (2007).
[00135] An isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal site that is different from its natural chromosomal location.
[00136] An isolated nucleic acid encoding an antiCD47 antibody or antigen-binding fragment thereof refers to one or more nucleic acid molecules encoding heavy and light chains of the antibody (or antigen-binding fragment thereof), including the nucleic acid molecule (s) present in a single vector or separate vectors, and the nucleic acid molecule (s) present in one or more locations in a host cell.
[00137] The percentage (%) of amino acid sequence identity to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence identical to the amino acid residues in the reference polypeptide sequence, after alignment sequences and gaps, if necessary, to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining the percentage amino acid sequence identity can be obtained with various methods in the art, for example
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41/99 pio, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. A person skilled in the art can determine suitable parameters for measuring alignment, including any algorithms necessary to obtain maximum alignment over the entire length of the sequences in comparison.
[00138] When referring to percentages of sequence identity in the present application, those percentages are calculated over the entire length of the longest sequence, unless specifically stated otherwise. The calculation over the entire length of the longest sequence is applicable to both nucleic acid sequences and polypeptide sequences.
[00139] The terms red blood cell and RBC are synonymous and used interchangeably.
[00140] The term agglutination refers to cell aggregation, and the term hemagglutinating reaction refers to the agglomeration of a specific subset of cells (namely, red blood cells). Consequently, the hemagglutination reaction is a type of agglutination.
[00141] 1.2 ANTI-CD47 ANTIBODY OF THE INVENTION [00142] The terms integrin-associated protein (IAP) and CD47, when used in this document, refer to any CD47 native to any vertebrate source, including mammals, such as primates (for example, humans) and rodents (for example, mice and rats), unless otherwise indicated. The term encompasses complete, untransformed CD47, as well as any form of CD47 or any fragment that results from transformation in the cell. The term also includes naturally occurring variants of CD47, for example, splicing variants or allelic variants.
[00143] The terms anti-CD47 antibody, anti-CD47, CD47 antibody and an antibody that binds to CD47 refer to an antibody
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42/99 that is able to bind to the CD47 protein or a fragment thereof with sufficient affinity, so that the antibody can be used as a diagnosis and / or therapeutic agent in the selection of CD47. In one embodiment, the degree of binding of an anti-CD47 antibody to a protein other than unrelated CD47 is less than about 10% of the antibody's binding to CD47 as measured, for example, by a radioimmunoassay (RIA). In some embodiments, an antiCD47 antibody provided in this document has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM or <0.001 nM (for example, 10-8 M or below 10 ^-8 M, for example 10 ' ⁸ M to 10 ¹³ M, for example 10 ⁹ M to 10 ¹³ M).
[00144] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention comprises substitutions, insertions or deletions. In the preferred mode, substitutions, insertions or exclusions occur in regions outside the CDRs (for example, in the FRs). Alternatively, the anti-CD47 antibody of the invention comprises post-translational modifications in the variable region of the light chain, in the variable region of the heavy chain, of the light or heavy chain.
An anti-CD47 antibody provided in the invention exhibits inhibitory activity, for example, to inhibit CD47 expression (for example, inhibit CD47 expression on the surface of a cell), activity and / or signaling, or interfere with interaction between CD47 and SIRPcl An anti-CD47 antibody provided in the invention results in the expression or activity of CD47 totally or partially decreased or regulated, after binding or interaction with CD47 (e.g., human CD47). The biological function of CD47 is decreased or regulated totally, significantly or partially after interaction between the antibody and the human CD47 polypeptide and / or peptide. When the level of expression or activity of CD47 in the presence of an antibody here
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43/99 described is reduced by at least 95% (for example, 96%, 97%, 98%, 99% or 100%) compared to the level of expression or activity of CD47 in the absence of interaction with (for example, binding a) the antibody, the antibody is considered capable of completely inhibiting CD47 expression or activity. The level of expression or activity of CD47 in the presence of an anti-CD47 antibody described here is reduced by at least 50% (for example, by 55%, 60%, 75%, 80%, 85% or 90%) in comparison at the level of CD47 expression or activity in the absence of binding to the anti-CD47 antibody, the antiCD47 antibody is considered capable of significantly inhibiting the expression or activity of CD47. The level of expression or activity of CD47 in the presence of an antibody described here is decreased by less than 95% (for example, by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75% , 80%, 85% or 90%) compared to the level of expression or activity of CD47 in the absence of interaction with (for example, binding to) the antibody, the antibody is considered capable of partially inhibiting the expression or activity of CD47.
[00146] In some embodiments, one or more amino acid modifications can be introduced in the Fc region of an antibody provided here, thus generating a variant of the Fc region. The Fc region variant may include a human Fc region sequence (for example, a human IgG1, IgG2, IgG3, or human IgG4 Fc region) comprising an amino acid modification (for example, a substitution) at one or more amino acid positions .
[00147] In some embodiments, it may be desirable to create antibodies engineered with cysteine, for example, thioMAbs, in which one or more residues of an antibody are replaced with cysteine residues.
[00148] In some embodiments, an antibody provided in this document can be further modified to contain other portions not
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44/99 proteins that are known in the art and readily available. Suitable portions for antibody derivation include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrhoHdone, poly-1,3-dioxolane, poH- 1,3,6-tnoxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers , polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol and mixtures thereof.
[00149] In some embodiments, the invention comprises fragments of an anti-CD47 antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab ^! ) 2 bodies; linear antibodies; single chain antibody molecules (for example, scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment, whose name reflects its ability to readily crystallize. Treatment with pepsin produces an F (ab ') 2 fragment that has two antigen-combining sites and is still capable of cross-linking the antigen.
[00150] In some embodiments, the anti-CD47 antibody of the invention is a humanized antibody. Different methods for humanized antibodies are known to the person skilled in the art, for example, as reviewed by Almagro & Fransson, whose content is hereby incorporated by reference in its entirety (Almagro JC and Fransson J., (2008), Frontiers in Bioscience 13: 1619- 1633). Almagro & Fransson make up
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45/99 yielded from rational and empirical approaches. The rational approach is characterized by the generation of some engineered antibody variants and evaluation of their binding properties or any other properties of interest. If the variants do not have the expected effect, a new round of connection designation and evaluation will begin. The rational approach includes CDR grafting, Resurfacing, Super-humanization and Content Optimization of the Human Chain. In contrast, the empirical approach is based on the generation of a large library of humanization variants and the selection of ideal clones with enrichment or high-throughput screening techniques. Thus, the empirical approach is dependent on a reliable selection and / or screening system capable of searching against a large number of antibody variants. In vitro display technologies, for example, phage display and ribosome display, meet these requirements and are well known to those skilled in the art. The empirical approach includes RF library, Guided selection, Shuffling of structures and Humanization.
[00151] In some embodiments, the anti-CD47 antibody of the invention is a human antibody. Antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450459 (2008).
[00152] The antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, several methods are known in the art for generating phage display libraries and screening those libraries for antibodies that have the desired binding characteristics. These methods are, for example, reviewed in Hoogenboom et al., In: Methods in Molecular Biology 178: 1-37 (O’Brien, et al.,
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46/99
Ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty, et al, Nature 348: 552-554; Clackso et al, Nature 352: 624-628 (1991); Marks, et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in: Methods in Molecular Biology 248: 161-175 (Lo, Ed., Humana Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Know. USA (101) 34: 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284 (1-2): 119132 (2004).
[00153] An antibody and antigen-binding fragment thereof suitable for use in that invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heterogeneously conjugated, multispecific, heterogeneously hybridized, chimeric, humanized (especially grafted) antibodies with CDR), dehumanized or human, Fab fragments, Fab 'fragments, F (ab') 2 fragments, fragments produced from a Fab, Fd, Fv, disulfide-bound Fvs (dsFvs) expression library, chain antibodies single (for example, scFvs), antibodies or teribodies (Holliger P., et al (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), nanobodies (also called single domain antibodies) , anti-idiotypic (anti-ld) antibodies (including, for example, anti-ld antibodies to an antibody of the invention) and epitope-binding fragments of any of the above. [00154] In some embodiments, an antibody of the present invention can be monospecific, blespecific or multispecific. A multispecific monoclonal antibody can be specific for different epitopes on a target polypeptide or can contain antigen-binding domains specific for more than one of the target polypeptides. See, for example, Tutt et al., (1991) J. Immunol. 147: 60-69. An anti-CD47 monoclonal antibody can be linked to or coexpressed with another hand
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47/99 functional molecule (for example, another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (for example, by chemical coupling, genetic fusion, non-covalent association or others) to one or more other molecules, in order to create bl- or multispecific antibodies with the second specificity of connection or more.
[00155] In some embodiments, the antibody of the invention binds to the human CD47 protein.
[00156] 1.3 NUCLEIC ACIDS OF THE PRESENT INVENTION AND
HOSTING CELLS UNDERSTANDING THEM [00157] In one aspect, the invention provides nucleic acids that encode any of the anti-CD47 antibodies or fragments thereof mentioned above. Nucleic acids can encode an amino acid sequence comprising the variable regions of the light and / or heavy chain of an antibody, or an amino acid sequence comprising the light and / or heavy chains of an antibody [00158] In one embodiment, a or more vectors comprising nucleic acids. In one embodiment, the vector is an expression vector.
[00159] In one embodiment, a host cell comprising the vector is provided. Host cells suitable for cloning or expression of vectors encoding antibodies include the eukaryotic or prokaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and the Fc effector function are not necessary. For expression of antibody and polypeptide fragments in bacteria, see, for example, US Patent Nos. 5,648,237, 5,789,199 and 5,840,523; and also Charlton, in: Methods in Molecular Biology, Volume 248 (Lo,
B.K.C. (Ed.), Humana Press, Totowa, NJ (2003), pages 245-254, which describe the expression of antibody fragments in E. coli). After
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48/99 expression, the antibody can be isolated from the bacterial cell mass in a soluble fraction and can be further purified.
[00160] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of yeast cells, mammalian cells or other cells that are suitable for preparing the antibodies or their antigen-binding fragments. For example, eukaryotic microbes, such as yeasts or filamentous fungi, are the appropriate expression or cloning hosts for vectors encoding antibodies, including strains of fungi and yeasts, whose glycosylation pathways have been humanized, resulting in the production of an antibody with a pattern glycosylation totally or partially human. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li, et al., Nat. Biotech. 24: 210-215 (2006). Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 (COS-7) cell lines transformed with SV40; embryonic human kidney cells (293 or 293T cells, as described, for example, in Graham, et al., J. Gen Virol. 36: 59 (1977)). Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO), including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, in: Methods in Molecular Biology, Volume 248 (B.K.C. Lo, (Ed.), Humana Press,
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49/99
Totowa, NJ) pages 255-268 (2003).
[00161] In one embodiment, a method of preparing an anti-CD47 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as indicated above, under conditions suitable for expression of the antibody, and optionally recovering the host cell antibody (or host cell culture medium). For recombinant production of an anti-CD47 antibody, nucleic acid encoding an antibody, for example, the antibody described above, is isolated and inserted into one or more vectors for cloning and / or expression in a host cell. This nucleic acid can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to genes encoding the antibody's heavy and light chains).
[00162] 1.4 PHARMACEUTICAL COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS [00163] The present invention further provides pharmaceutical compositions comprising one or more monoclonal antibodies that bind to CD47 or its immunologically active fragment. It is to be understood that the anti-CD47 antibody or pharmaceutical composition provided for in the present invention can be formulated in vehicles, excipients and other suitable agents in a formulation for co-administration, resulting in better transfer, delivery, tolerance and the like.
[00164] The term pharmaceutical composition refers to a formulation that is present in a form so as to allow the biological activity of the active ingredient contained in the formulation to be effective, and that it does not contain additional components that are unacceptably toxic to an individual to whom the formulation would be administered.
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50/99 [00165] The term pharmaceutical vehicle refers to diluents, adjuvants (for example, Freund's adjuvant (complete or incomplete)), excipients or vehicles with which the therapeutic agent is administered.
[00166] For use in this document, treatment refers to relieving, interrupting, delaying, alleviating, ceasing, reducing or reversing the progression or severity of the existing symptom, disorder, condition or disease, and preventing the relapse of the relevant disease.
[00167] In some embodiments, the invention comprises an anti-CD47 monoclonal antibody conjugated to a therapeutic module, such as a cytotoxic agent or an immunosuppressive agent (immunoconjugate ”). Cytotoxic agents include any of the harmful drugs for cells. Examples of suitable cytotoxic agents for forming an immunoconjugate (for example, chemotherapeutic agents) are known in the field. See, for example, Document WO 05/103081. For example, cytotoxic agents include, but are not limited to: Radioactive Isotopes (for example, At ²¹¹ , I ¹³¹ , I ¹²⁵ , γ ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , pb ²¹² and isotopes radioactive substances); chemotherapeutic agents or drugs (for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or their variants; and the various known anti-tumor or anti-cancer agents.
The present invention further includes a composition comprising an anti-CD47 antibody (including a pharmaceutical composition or pharmaceutical formulation) and a composition comprising
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51/99 giving a polynucleic acid that encodes the anti-CD47 antibody. In some embodiments, the composition comprises one or more antibodies that bind CD47 or one or more polynucleic acids that encode one or more antibodies that bind CD47. Such compositions may further comprise suitable pharmaceutical carriers, such as pharmaceutical excipients known in the field, including buffering agents.
[00169] The pharmaceutical compositions of the invention can comprise an antibody of the invention and pharmaceutical carriers. This pharmaceutical composition can be contained in a kit, such as a diagnostic kit.
[00170] Pharmaceutical vehicles suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical carriers include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skimmed milk, glycerol, propylene glycol, water, ethanol and the like. For the use of excipients and their uses, see also Handbook of Pharmaceutical Excipients, 5th Edition, Rowe RC; PJ Seskey and SC Owen, Pharmaceutical Press, London, Chicago. The compositions, if desired, can also contain small amounts of moisturizing or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. THE
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52/99 oral formulation may include conventional vehicles, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate and saccharin.
[00171] Pharmaceutical formulations comprising an anti-CD47 antibody of the invention described herein may be prepared by mixing ~ the anti-CD47 antibody of the invention having a degree of purity with optional one or more pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980), preferably in the form of lyophilized formulations or aqueous solutions.
Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and Document W02006 / 044908, the latter formulations including a histidine-acetate buffer.
[00173] The pharmaceutical compositions or formulations of the invention may further contain more than one active ingredient, as necessary for the particular indication being treated, preferably those with complementary activities without mutual harmful effect. For example, statins are also ideally supplied. The active ingredients are present properly in combination, in amounts effective for the use of interest.
[00174] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, whose matrices are shaped articles, for example, films or microcapsules.
[00175] 1.5 THERAPEUTIC METHODS WITH ANTIBODIES AND
ITS USE [00176] In one aspect, the present invention is directed to a method of inhibiting and / or antagonizing the binding of CD47 to SIRPa in a
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53/99 individual, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments thereof described herein. In another aspect, the present invention is directed to a method for facilitating phagocytosis of phagocytic cells in an individual, comprising administering to the individual the effective amount of any of the anti-CD47 antibodies or fragments described herein. In one aspect, the present invention is directed to a method of treating the relevant disease with CD47 being the therapeutic target, comprising administering to an individual the effective amount of any of the anti-CD47 antibodies or fragments described herein. In one aspect, the present invention is directed to a method for any disease or disorder that can be ameliorated, delayed, inhibited or prevented by eliminating, inhibiting or decreasing the binding of CD47 to SIRPa. In another aspect, the present invention provides methods of treating a cancer or tumor in an individual in need, to alleviate symptoms of cancer or tumor in the individual, and to prevent recurrence of the cancer or tumor in the individual, by administering to the individual of the anti-CD47 antibody or fragments of the invention.
[00177] In one aspect, the anti-CD47 antibody, the antigen-binding fragment thereof and the pharmaceutical composition comprising what are provided for in the present invention can be used as a therapeutic agent for diagnosis, prognosis, monitoring, treatment, attenuation and / or prophylaxis of a disease and disorder relevant to the aberrant CD47 expression, activity and / or signaling in an individual. By identifying the presence of the disease and disorder relevant to the aberrant CD47 expression, activity and / or signaling in an individual with a standard assay, the anti-CD47 antibody, the antigen binding fragment and the pharmaceutical composition can be administered understanding them.
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In other respects, the present invention provides for the use of anti-CD47 antibody in the manufacture or preparation of a medicament to treat the aforementioned disease or relevant disorder.
[00179] In some embodiments, the method and use described herein further include administering to said individual the effective amount of at least one additional therapeutic agent, for example, a chemotherapeutic agent, radiotherapeutic agent or biomacromolecular drug. In one embodiment, the biomacromolecular drug is, for example, several monoclonal antibody drugs that attack tumor cells through recognition by T cells, for example, rituximab, cetuximab and trastuzumab.
The combination therapy mentioned above comprises the combined administration (in which more than two of the therapeutic agents are included in the same formulation or individual formulations) and the isolated administration, in which the administration of the anti-CD47 antibody of the invention can occur before, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvants.
[00181] An antibody according to the invention (and any of the additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administrations and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be via an appropriate route, through injection, for example, subcutaneous or intravenous injection, depending on the short-term or long-term nature of the administration to a certain extent. Various dosage regimens are contemplated in this document, including, but not limited to, single or multiple administrations over several times, bolus and infusion administration
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Pulsed 55/99.
[00182] For the prevention or treatment of a disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is adequately administered to the patient either once or over a series of treatments.
[00183] In another aspect, the antibody of the invention may be useful in detecting the course of a therapy for CD47-related disease in vivo or in vitro. For example, it can be determined whether a particular therapy to treat disease or alleviate symptoms is effective or not by measuring the increased or decreased number of cells that express CD47 (for example, cancer cells).
[00184] Most anti-CD47 antibodies are reported to induce the hemagglutination reaction of human red blood cells. Hemagglutination is an example of homotypic interaction, in which treatment with a divalent CD47-binding entity induces the aggregation or agglutination of two cells that express CD47. For example, an anti-CD47 MABL antibody such as a total IgG or F (ab ') 2 has been reported to allow the red blood cell hemagglutination reaction, and the effect was weakened only if the MABL was altered in a scFv or a bivalent scFv (for example, Uno S, Kinoshita Y, Azuma Y, et al., Antitumor activity of a monoclonal antibody against CD47 in xenograft models of human leukemia, Oncol Rep 2007; 17: 1189-94; Kikuchi Y, Uno S, Yoshimura Y , et al., A bivalent singlechain Fv fragment against CD47 induces apoptosis for leukemic cells, Biochem Biophys Res Commun 2004; 315: 912-8). The other antibodies
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56/99 known anti-CD47 wells (including B6H12, BRC126 and CC2C6) can also result in a RBC hemagglutination reaction. Therefore, the agglutination of cells is the main limitation regarding the therapeutic selection of CD47 with the total existing IgG antibodies.
[00185] Considering that most of the antibodies described in the technique that block the interaction of CD47 with SIPRa to facilitate phagocytosis will result in apparent cell agglutination, there is still a great need for new anti-CD47 antibodies that are not only capable of effectively facilitating phagocytosis by macrophages, but which also do not lead to cell agglutination. In this regard, the need is met by the anti-CD47 antibodies described in the present patent application, which not only can be effective in facilitating phagocytosis, even exert excellent effects of antitumor growth and tumor elimination, but also do not result in apparent agglutination of cells, while exerting therapeutic effects, thus having significantly reduced side effects.
[00186] One skilled in the art can quantify the level of agglutination with conventional experience, for example, the RBC hemagglutination reaction. For example, a hemagglutination test can be performed by a person skilled in the art in the presence of the anti-CD47 antibody of the invention, followed by measuring the area of the RBC points to determine the level of the hemagglutination reaction, as described in the examples below. In some cases, the comparison was made between the areas of the RBC points in the presence of the anti-CD47 antibody of the invention and in the absence of the anti-CD47 antibody of the invention (namely, under a condition of zero hemagglutination reaction), as well as in the presence of other known anti-CD47 antibodies. Thus, the hemagglutination reaction was quantified against the baseline control. The larger the area of the CBR points, the greater the
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57/99 level of the hemagglutination reaction. Alternatively, the hemagglutination reaction can also be quantified by analyzing the density of the RBC points.
[00187] 1.6 METHODS AND COMPOSITIONS FOR DIAGNOSIS
AND DETECTION [00188] In some embodiments, any of the anti-CD47 antibodies or their antigen-binding fragments provided here is useful for detecting the presence of CD47 in a biological sample. The term detection, as used herein, includes quantitative or qualitative detection. In some embodiments, a biological sample is blood, serum or other liquid samples from biological sources. In some embodiments, a biological sample comprises a cell or tissue.
[00189] In some embodiments, labeled anti-CD47 antibodies are provided. Markers include, but are not limited to, markers or portions that are detected directly (such as fluorescent, chromophores, electron-dense, chemiluminescent and radioactive markers), as well as portions, such as enzymes or ligands, that are detected indirectly, for example, through an enzymatic reaction or molecular interaction. Exemplary markers include, but are not limited to, the radioisotopes ³² P, ¹⁴ C, ¹²⁵ l, ³ H, and ¹³¹ l, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansila, umbeliferone, luceriferases , for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazinedione, horseradish (HR) peroxidase, alkaline phosphatase, β-galactosldase, glycoamylase, lysozyme, saccharide oxidases , for example, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that uses hydrogen peroxide to oxidize a dye precursor, such as HR, lactoperoxidase or microperoxidase; biotin / avidin, brand
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58/99 rotation pain, bacteriophage markers, stable free radicals and the like.
[00190] The invention is further illustrated by the following examples. However, it is to be understood that the invention is described with the examples in an illustrative rather than limited way, and various modifications can be made by the person skilled in the art.
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1.7 SEQUENCES OF ANTI-CD47 ANTIBODIES EXAMPLES OF THE INVENTION
TABLE A. SEQUENCES OF LIGHT AND HEAVY CHAIN CDRS OF THE EXEMPLIFICATIVE ANTIBODIES OF THE INVENTION
ADI Name CDR1 VH CDR2VH CDR3 VH CDR1 VL CDR2VL CDR3VL Position byNumberingKabat H27-35 H50-65 H93-102 L24-34 L50-56 L89-97 ADI-26624 GSISSYYWS (SEQ IDNO: 1) YIYYSGSTNYNPSLKS (SEQ ID NO: 9) ARGKSAFDP (SEQ ID NO: 17) RASQGISRWLA(SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQADLHPPLT (SEQ ID NO: 27) ADI-29336 GSISNYYWS (SEQ IDNO: 2) TIYYSGSTRYNPSLKS (SEQ ID NO: 10) ARGKSAFNP (SEQ ID NO: 18) RASQGISRWLA (SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQADLHPPLT (SEQ ID NO: 27) ADi-29340 GSIDYYYWS (SEQ IDNO: 3) YIYYSGSTGYNPSLKS (SEQ ID NO: 11) ARGKSAFDP (SEQ ID NO: 17) RASQGISRWLA (SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQADLHPPLT (SEQ ID NO: 27) ADI-26630 GSISSYYWS (SEQ IDNO: 1) YIYYSGSTNYNPSLKS (SEQ ID NO: 9) ARGKTGSAA (SEQ ID NO: 19) RASQGISRWLA (SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQTVSFPIT (SEQ ID NO: 28) ADI-29341 GSIEHYYWS (SEQ IDNO: 4) YIYYSGSTNYNPSLKS (SEQ ID NO: 9) ARGKTGSAA (SEQ ID NO: 19) RASQGISRWLA (SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQTVSFPIT (SEQ ID NO: 28) ADI-29349 GSIDHYYWS (SEQ ID NO: 5) YiYYSGSTEYNPSLKS (SEQ ID NO: 12) ARGKTGSAA (SEQ ID NO: 19) RASQGISRWLA (SEQ ID NO: 23) AASSLQS (SEQ ID NO: 25) QQTVSFPIT (SEQ ID NO: 28)GSIX1X2YYWS (where X1 is selected from the group consisting of S, D or E; and X2 is selected from the group consisting of S, N, Y or XIIYYSGSTX2YNPSLKS, where XI is selected from the group consisting of Y or T; and X2 is selected from the group consisting of N, R, G or N (SEQ ID NO: 100) ARGKX1X2X3X4X5, where X1 is selected from the group consisting of S or T; X2 is selected from the group consisting of A or G; X3 is selected from the group consisting of
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ADS Name CDR1 VH CDR2VH CDR3 VH CDR1 VL CDR2VL CDR3VLH) (SEQ ID NO: 98)in F or S; X4 is selected from the group consisting of D, N or A, and X5 is selected from the group consisting of P or A (SEQ ID NO: 102) ADI-26591 FTFSSYAMS (SEQ ID NO: 6) AISGSGGSTYYADSVKG (SEQ ID NO: 13) AKTPIYYGFDL (SEQ ID NO: 20) RASQGISSWLA (SEQ ID NO: 24) GASSLQS (SEQ ID NO: 26) QQKNPFPPT (SEQ ID NO: 29) ADI-29371 FTFGNYAMS (SEQ IDNO: 7) MISG-GGSTYYADSVKG(SEQ ID NO: 14) AKTPIYYGFDL (SEQ ID NO: 20) RASQGISSWLA (SEQ ID NO: 24) GASSLQS (SEQ ID NO: 26) QQKNPFPPT (SEQ ID NO: 29) ADI-30793 FTFDSYAMT (SEQ IDNO: 8) VISGSGGKTYYADSVKG(SEQ ID NO: 15) AKTHLYYGFDL (SEQ ID NO: 21) RASQGISSWLA (SEQ ID NO: 24) GASSLQS (SEQID NO: 26) QQKNPFPPF (SEQ ID NO: 30) ADI-30794 FTFGNYAMS (SEQ IDNO: 7) AISGSGGKTYYADSVKG (SEQ ID NO: 16) AKTAIYYGFDL (SEQ ID NO: 22) RASQGISSWLA (SEQ ID NO: 24) GASSLQS (SEQ ID NO: 26) QQKNPFPPF (SEQ ID NO: 30)FTFX1X2YAMX3 (where X1 is selected from the group consisting of S, G or D; X2 is selected from the group consisting of S or N; and X3 is selected from the group consisting of S or T (SEQ ID NO: 99) XIISGX2GGX3TYYADSVKG, where X1 is selected from the group consisting of A, M or V; X2 is selected from the group consisting of S or exclusion; and X3 is selected from the group consisting of S or K (SEQ ID NO: 101) AKTX1X2YYGFDL, where X1 is selected from the group consisting of P, H or A; and X2 where X1 is selected from the group consisting of I or L (SEQ ID NO: 103)
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TABLE B. SEQUENCES OF VARIABLE REGIONS OF LIGHT AND HEAVY CHAINS OF ANTIBODIES
EXAMPLIFICATIONS OF THE INVENTION
Name DNA VH VH protein DNA VL VL protein ADI-26624 CAGG! GCAGC! GCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGAGTGCATTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 59) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGKSAFDPWGQGTLVTVSS (SEQ ID NO: 44) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCAGACCTCCACCCTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQGISRWLA WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQADLHPPLTFGG GTKVEIK (SEQ ID NO: 54) ADI-29336 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGACGATCTATTACAGTGGGAGCACCCGTTACAACCCCTCCCTCAAGAGTC QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPPGKGLEWIGTIYYSGSTRYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGKSAFNPWGQGTLVTVSS (SEQ ID NO: 45) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADLHPPLTFGGGTKVEIK (SEQ ID NO:
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Name DNA VH VH protein DNA VL VL proteinGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGAGTGCATTCAACCCATGGGGACAGGGTACATTGGTCACCGT CTCCTCA (SEQ ID NO: 60)AGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCAGACCTCCACCCTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 69) 54) ADI-29340 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCGATTATTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACTCGGGGAGCACCGGTTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGAGTGCATTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 61) QVQLQESGPGLVKPSETLSLTCTVSGGSIDYYYWSWIRQPPGKGLEWIGYIYYSGSTGYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV YYCARGKSAFDPWGQGTLVTVSS (SEQ ID NO: 46) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGCAGACCTCCACCCTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 69) DIQMTQSPSSVSASVGD RVTITCRASQGISRWLA WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTiSSLQPEDFAT YYCQQADLHPPLTFGG GTKVEIK (SEQ ID NO: 54) ADI-26630 CAGGT GCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCC QVQLQESGPGLVKPSETLSLTCTVSGGSiSSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISV GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCC DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS
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Name DNA VH VH protein DNA VL VL proteinGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGACGGGATCTGCCGCATGGGGACAGGGTACATTGGTCACCGT CTCCTCA (SEQ ID NO: 62) DTSKNQFSLKLSSVTAADTAVYYCARGKTGSAAWGQGTLVTVSS (SEQ ID NO: 47) CTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGACAGTCTCCTTCCCTATCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 70) GTDFTLTISSLQPEDFATYYCQQTVSFPITFGGGTKVEIK (SEQ ID NO: 55) ADI-29341 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCGAGCATTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGACGGGATCTGCCGCATGGGGACAGGGTACATTGGTCACCGT CTCCTCA (SEQ ID NO: 63) QVQLQESGPGLVKPSETLSLTCTVSGGSIEHYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGKTGSAAWGQGTLVTVSS (SEQ ID NO: 48) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGACAGTCTCCTTCCCTATCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 71) DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVSFPITFGGGTKVEIK (SEQ ID NO: 55) ADI- CAGGTGCAGCTGCAGGAGTCGGGCCCA QVQLQESGPGLVKPSETL GACATCCAGATGACCCAGTCTCCATCTTCCGTG DIQMTQSPSSVSASVGD
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Name DNA VH VH protein DNA VL VL protein 29349 GGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCGATCATTACTACTGGAGTTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACTCTGGGAGCACCGAGTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGGGGTAAGACGGGATCTGCCGCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 64) SLTCTVSGGSIDHYYWSWIRQPPGKGLEWIGYIYYSGSTEYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGKTGSAAWGQGTLVTVSS (SEQ ID NO: 49) TCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGACAGTCTCCTTCCCTATCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 70) RVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVSFPITFGGGTKVEIK (SEQ ID NO: 55) ADI-26591 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGACGCCTATATACTACGG EVQLLESGGGLVQPGGSL RLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTPIYYGFDLWGRGTLVTVSS (SEQ ID NO: 50) GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA Illi GCAACTTATTACTGTCAGCAGAAAAATCCCTTCCCTCCTACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ IDNO: 72) DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKNPFPPTFGGGTKVEIK (SEQ ID NO: 57)
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Name DNA VH VH protein DNA VL VL proteinCTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 65) ADI-29371 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGGGAATTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCAATGATTAGTGGGGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGACGCCTATATACTACGGCTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 66) EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSWVRQAPGKGLEWVSMISGGGSTYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKTPIYYGFDLWGRGTLVTVSS (SEQ ID NO: 51) GACATCCAGTTGACCCAGTCTCCATCTTCCGTG TCTGCATCTGTAGGAGACAGAGTCACCATCACT TGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCC CTAAGCTCCTGATCTATGGTGCATCCAGTTTGC AAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTAC TGTCAGCAGAAAAATCCCTTCCCTCCTACTTTTG GCGGAGGGACCAAGGTTGAGATCAAA (SEQ IDNO: 72) DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKNPFPPTFGGGTKVEIK (SEQ ID NO: 57) ADI-30793 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATAGCTATGCCATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTAGTGGAAGTGGTGGTAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACTCCAA EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMTWVRQAPGKGLEWVSVISGS GGKTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAKTHLYYGFDLW GRGTLVTVSS (SEQ ID NO: 52) GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTAC DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKNPFPPFFGGGTKVEIK (SEQ ID NO: 58)
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Name DNA VH VH protein DNA VL VL proteinGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGACGCATCTTTACTACGGCTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 67)TGTCAGCAGAAAAATCCCTTCCCTCCTTTTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 73)ADI-30794 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGGGAATTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGAAGTGGTGGTAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGACGGCTATATACTACGGCTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 68) EVQLLESGGGLVQPGGSL RLSCAASGFTFGNYAMSWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKTAIYYGFDL WGRGTLVTVSS (SEQ ID NO: 53) GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGAAAAATCCCTTCCCTCCTTTTTTTGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 73) DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTiSSLQPEDFATYYCQQKNPFPPFFGGGTKVEIK (SEQ ID NO: 58)
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TABLE C. FRS SEQUENCES OF LIGHT AND HEAVY CHAINS OF EXEMPLIFICATIVE ANTIBODIES
THE INVENTION
First name FR1 VH FR2 VH FR3 VH FR4 VH FR1 VL FR2 VL FR3 VL FR4VL ADI-26624, ADI-29336, ADI-29340, ADI-26630, ADI-29341, ADI-29349 QVQLQESGPGLVKPSETLSLTCTVSG (SEQ ID NO: 31) WIR-QPPGKGLEWIG (SEQID NO: 33) RVTISV-DTSKNQFSL-KLSSVTAADTAVYYC (SEQID NO: 35) WGQG-TLVTVSS(SEQ IDNO: 37) DIQMTQSPSSVSAS VGDRVTITC (SEQ ID NO: 39) WYQQK-PGKAPKLLIY (SEQ ID NO: 41) GVPS-RFSGSGSG-TDFTLTISS-LQPEDFATYYC(SEQ ID NO: 42) FGGGT-KVEIK (SEQID NO: 43) ADI-26591, ADI-29371, ADI-30793, ADI-30794 EVQLLESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 32) WVR-QAPGKGLEWVS (SEQID NO: 34) RFTIS-RDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 36) WGRG-TLVTVSS(SEQ IDNO: 38) DIQLT-QSPSSVSASVGDRVTITC(SEQ ID NO:40) WYQQK-PGKAPKLLIY (SEQ ID NO: 41) GVPS-RFSGSGSG-TDFTLTISS-LQPEDFATYYC(SEQ ID NO: 42) FGGGT-KVEIK (SEQID NO: 43)
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TABLE D. NUMBERING OF A PART OF THE SEQUENCES IN THIS SEQUENCE LISTING
NameADI SEQ iD NO for the heavy chain SEQ ID NO for the ieve chain IgG4 igG1 VH heavy chain variable region Variable region of the ieve chainFR1 VH CDR1VH FR2VH CDR2 VH FR3 VH CDR3 VH FR4 VH DNAVH Pro.VH FR1VL CDR1 VL FR2 VL CDR2 VL FR3VL CDR3 VL FR4VL DNAVL Pro.VL HC'sJailHeavy LC ofHere-hateLight , I LC of i _ HC. . | Here-Chain ί,. „. i goHeavy | ,| Light ADI-26624 31 1 33 9 35 17 37 59 44 39 23 41 25 42 27 43 69 54 74 75 88 75 ADi29336 31 2 33 10 35 18 37 60 45 39 23 41 25 42 27 43 69 54 76 75 89 75 ADi29340 31 3 33 11 35 17 37 61 46 39 23 41 25 42 27 43 69 54 77 75 90 75 ADI26630 31 1 33 9 35 19 37 62 47 39 23 41 25 42 28 43 70 55 78 79 91 79 ADI29341 31 4 33 9 35 19 37 63 48 39 23 41 25 42 28 43 71 55 80 79 92 79 ADi29349 31 5 33 12 35 19 37 64 49 39 23 41 25 42 28 43 70 55 81 79 93 79 ADi-26591 32 6 34 13 36 20 38 65 50 40 24 41 26 42 29 43 72 56 82 83 94 83 ADI29371 32 7 34 14 36 20 38 66 51 40 24 41 26 42 29 43 72 57 84 83 95 83 ADi30733 32 6 34 15 36 21 38 67 52 40 24 41 26 42 30 43 73 58 85 88 96 86 ADi-30794 32 7 34 16 36 22 38 68 53 40 24 41 26 42 30 43 73 58 67 86 97 86
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69/99 [00191] COMPLETE AMINO ACID SEQUENCE OF HEAVY CHAIN ANTIBODIES AND LIGHT OF THE INVENTION [00192] ADI26624-IgG4 [00193] amino acid sequence of HC (SEQ ID NO: 74) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY CARGKSAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLG [00194] LC Amino Acid Sequence (SEQ ID NO: 75)
DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADLHP
PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC [00195] SEQ-436]
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPPGKGLE WIGTIYYSGSTRYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKSAFNPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
Petition 870190067389, of 7/17/2019, p. 173/216
70/99
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLG [00197] LC Amino Acid Sequence (SEQ ID NO: 75) [00198] ADI29340-lgG4 [00199] HC Amino Acid Sequence (SEQ ID NO: 77)
QVQLQESGPGLVKPSETLSLTCTVSGGSIDYYYWSWIRQPPGKGLE
WIGYIYYSGSTGYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKSAFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLG [00200] LC Amino Acid Sequence (SEQ ID NO: 75) [00201] ADI26630-lgG4 [00202] HC Amino Acid Sequence (SEQ ID NO: 78)
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE
WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLG [00203] LC Amino Acid Sequence (SEQ ID NO: 79)
DIQMTQSPSSVSASVGDRVTiTCRASQGISRWLAWYQQKPGKAPKLL
Petition 870190067389, of 7/17/2019, p. 174/216
71/99
IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVSFP
ITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC [00204] ADI29341-lgG4 [00205] HC amino acid sequence (SEQ ID NO: 80)
QVQLQESGPGLVKPSETLSLTCTVSGGSIEHYYWSWIRQPPGKGLE
WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLG [00206] LC Amino Acid Sequence (SEQ ID NO: 79) [00207] ADI 29349 ~ lgG4 [00208] HC Amino Acid Sequence (SEQ ID NO: 81)
QVQLQESGPGLVKPSETLSLTCTVSGGSIDHYYWSWIRQPPGKGLE
WIGYIYYSGSTEYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLG [00209] LC Amino Acid Sequence (SEQ ID NO: 79)
Petition 870190067389, of 7/17/2019, p. 175/216
72/99 [00210] ADI 26591-lgG4 [00211] HC amino acid sequence (SEQ ID NO: 82)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTPIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG [00212] LC Amino Acid Sequence (SEQ ID NO: 83)
DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLI
YGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKNPFP
PTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC [00213] ADI 29371-lgG4 [00214] HC amino acid sequence (SEQ ID NO: 84)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSWVRQAPGKGL
EWVSMISGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKTPIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAA
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG
LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN
VFSCSVMHEALHNHYTQKSLSLSLG
Petition 870190067389, of 7/17/2019, p. 176/216
73/99 [00215] LC Amino Acid Sequence (SEQ ID NO: 83) [00216] ADI 30793-lgG4 [00217] HC Amino Acid Sequence (SEQ ID NO: 85)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMTWVRQAPGKGL
EVWSVISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTHLYYGFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG [00218] LC Amino Acid Sequence (SEQ ID NO: 86)
DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLI
YGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKNPFP
PFFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC [00219] ADI 30794-lgG4 [00220] HC amino acid sequence (SEQ ID NO: 87)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSVWRQAPGKGL
EVWSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTAIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNK
GLPSSÍEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
Petition 870190067389, of 7/17/2019, p. 177/216
74/99
NVFSCSVMHEALHNHYTQKSLSLSLG [00221] LC Amino Acid Sequence (SEQ ID NO: 86) [00222] ADI26624-lgG1 [00223] HC Amino Acid Sequence (SEQ ID NO: 88)
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE
WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKSAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00224] LC Amino Acid Sequence (SEQ ID NO: 75) [00225] ADI29336-lgG1 [00226] HC Amino Acid Sequence (SEQ ID NO: 89)
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQPPGKGLE
WIGTIYYSGSTRYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKSAFNPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00227] LC Amino Acid Sequence (SEQ ID NO: 75) [00228] ADI29340-lgG1 [00229] HC Amino Acid Sequence (SEQ ID NO: 90)
Petition 870190067389, of 7/17/2019, p. 178/216
75/99
QVQLQESGPGLVKPSETLSLTCTVSGGSIDYYYWSWIRQPPGKGLE
WIGYIYYSGSTGYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKSAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNVVYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00230] LC Amino Acid Sequence (SEQ ID NO: 75) [00231] ADI26630-lgG1 [00232] HC Amino Acid Sequence (SEQ ID NO: 91)
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE
WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00233] LC Amino Acid Sequence (SEQ ID NO: 79) [00234] ADI29341-lgG1 [00235] HC Amino Acid Sequence (SEQ ID NO: 92)
QVQLQESGPGLVKPSETLSLTCTVSGGSIEHYYWSWIRQPPGKGLE
WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
Petition 870190067389, of 7/17/2019, p. 179/216
76/99
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00236] LC Amino Acid Sequence (SEQ ID NO: 79) [00237] ADI 29349-lgG1 [00238] HC Amino Acid Sequence (SEQ ID NO: 93)
QVQLQESGPGLVKPSETLSLTCTVSGGSIDHYYWSWIRQPPGKGLE
WIGYIYYSGSTEYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYY
CARGKTGSAAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG [00239] LC Amino Acid Sequence (SEQ ID NO: 79) [00240] ADI 26591 ~ lgG1 [00241] HC Amino Acid Sequence (SEQ ID NO: 94)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSVWRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTPIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
Petition 870190067389, of 7/17/2019, p. 180/216
77/99
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG [00242] LC Amino Acid Sequence (SEQ ID NO: 83) [00243] ADI 29371-lgG1 [00244] HC Amino Acid Sequence (SEQ ID NO: 95)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSWVRQAPGKGL
EWVSMISGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKTPIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPG [00245] LC Amino Acid Sequence (SEQ ID NO: 83) [00246] ADI 30793-lgG1 [00247] HC Amino Acid Sequence (SEQ ID NO: 96)
EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMTVWRQAPGKGL
EVWSVISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTHLYYGFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPG [00248] LC Amino Acid Sequence (SEQ ID NO: 86) [00249] ADI 30794-lgG1
Petition 870190067389, of 7/17/2019, p. 181/216
78/99 [00250] HC amino acid sequence (SEQ ID NO: 97) EVQLLESGGGLVQPGGSLRLSCAASGFTFGNYAMSWVRQAPGKGL EVWSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAKTAIYYGFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKPPCD
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG [00251] LC amino acid sequence (SEQ ID NO: 86) [00252] CD47 protein amino acid sequence (SEQ ID NO: 56)
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNME
AQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKG
DASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNE
NILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVG AILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQV
IAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE [00253] The negative control sequences in the attached drawings are as follows:
[00254] lgG1 HC:
MGWSLILLFLVAVATRVLSEVRLLESGGGLVQPGGSLRLSCAASGFT
FSNYAMGVWRQAPGKGLEVWSAISGSGGSTYYADSVKGRFTTSRD DSKNALYLQMNSLRAEDTAVYYCARGGPGWYAADVWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYNSPE
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
Petition 870190067389, of 7/17/2019, p. 182/216
79/99
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQLNGS
MDFQVQIISFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCRASQ SISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [00256] IgG4 HC: MGWSLILLFLVAVATRVLSEVRLLESGGGLVQPGGSLRLSCAASGFT FSNYAMGWVRQAPGKGLEVWSAISGSGGSTYYADSVKGRFTTSRD DSKNALYLQMNSLRAEDTAVYYCARGGPGWYAADVWGQGTTVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [00257] IgG4 LC:
MDFQVQIISFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCRASQ SISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [00258] The negative control IgG1 class is used when the class IgG 1 test antibody are used; and Class lgG4 negative control is used when Class lgG4 test antibodies are used.
Petition 870190067389, of 7/17/2019, p. 183/216
80/99
EXAMPLES
EXAMPLE 1. PRODUCTION AND PURIFICATION OF AN ANTI-CD47 ANTIBODY AND THE CONTROL ANTIBODY [00259] In the sequence listing section of this application, the amino acid sequences of the CDR regions, the variable regions of the light and heavy chains, and the light and heavy chains of 10 antibodies exemplified in the present invention (ADI-26624, ADI-29336, ADI-29340, ADI-26630, ADI-29341, ADI-29349, ADI-26591, ADI-29371, ADI-30793, ADI- 30794), as well as the corresponding nucleotide sequences. In addition, sequence numbering for light and heavy chains, heavy chains, variable regions of the light and heavy chains of the exemplary antibodies mentioned above in the present invention is shown in Table 1.
[00260] The antibody of the invention was expressed in yeast cells and CHO-S cells, and purified.
EXPRESSION AND PURIFICATION IN YEAST CELLS [00261] Yeast-based antibody presentation libraries (Adimab) have been expanded according to existing procedures (W02009036379; WO 2010105256; W02012009568), with the diversity in each library being 1 χ 10 ⁹ . In summary, the classification of the activated magnetic cells was carried out with the Miltenyi Company's MACS system in the first two rounds of screening. First, the yeast cells in the libraries (~ 1x10 ¹ cells / library) were incubated separately in a FACS wash buffer (a phosphate buffer containing 0.1% of the whey protein) containing 100 nM of the CD47 antigen labeled with biotin (Acro Biosystems, Catalogs No. CD7-H5227-1 mg) at room temperature for 15 min. The cells were washed with 50 ml of the pre-cooled FACS wash buffer, then resuspended in 40 ml of the same wash buffer, and incubated at 4 ° C for 15 min after addition
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81/99 of 500 μΙ of microspheres of Streptinomycin (Miltenyi LS). Then, the supernatant was discarded by centrifugation at 1000 rpm for 5 min, cell pellets were resuspended in 5 ml of the FACS wash buffer and the cell suspension was loaded onto the Miltenyi LS column. After loading was complete, the column was washed with FACS wash buffer 3 times with 3 ml for each wash. The Miltenyi LS column was removed from the magnetic region and eluted with 5 ml of the growth medium. The eluted yeast cells were harvested and cultured overnight at 37 ° C.
[00262] A flow cytometer was used for the next round of classification: Approximately 1x10 ⁸ of yeast cells obtained through screening with the MACS system were washed with FACS buffer three times, and cultured in a culture broth containing labeled CD47 antigen. with biotin at a low concentration (100 ' ¹ nM) at room temperature. The culture broth was discarded, and the cells were washed with FACS wash buffer twice and then mixed with LC-FITC reagent (FITC-labeled goat antibody against the human immunoglobin F (ab ') cap chain) , Southern Biotech) (diluted 1: 100), and with reagent SA-633 (streptavidin-633, Molecular Probes) (diluted 1: 500) or SA-PE (streptavidin-phycoerythrin, Sigma) (diluted 1:50 ), incubated at 4 ° C for 15 min. The cells were eluted with the pre-cooled FACS wash buffer, centrifuged, resuspended in 0.4 ml of the buffer and transferred to a separation tube with a filter. The cells were classified with FACS ARIA (BD Biosciences).
[00263] Yeast cells obtained by screening, which expressed anti-CD47 antibodies, were induced at 30 ° C for 48 hours with agitation, to express antibodies against CD47. After the end of the induction, yeast cells were removed by centrifugation at 1300 rpm for 10 min and the supernatant collected. Antl antibodies
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CD47 in the supernatant were purified on a Protein A column, eluted with a pH 2.0 acetate solution. Anti-CD47 antibodies were harvested with> 95% antibody purity. The corresponding Fab fragments could be obtained by papain digestion and purification with KappaSelect (GE Healthcare Life Sciences).
EXPRESSION AND PURIFICATION IN CHO-S CELLS [00264] A CHO-S cell line expressing the antibody was established using the Freedom® CHO-S® kit (Invitrogen), according to the manufacturer's instructions. First, DNA sequences for the light and heavy chains of the antibody molecule were inserted into the same plasmid pCHO10, where the heavy chain is upstream of the light chain. The plasmid pCHOl.O constructed was then transferred to the CHO cell line with chemical transfection and electroporation. Antibody yield was detected using ForteBio 48 hours after transfection, to determine the efficiency of the transfection. A pool of cells with high antibody expression was obtained after the transfected cells were subjected to two rounds of selective screening. The cell pool was then propagated to express antibodies abundantly, and the cell supernatant was collected and purified on the Protein A column with antibody purity> 95%.
TABLE 1. NUMBERING FOR LIGHT AND HEAVY CHAIN AMINO ACID SEQUENCES, THE LIGHT AND HEAVY CHAIN VARIABLE REGIONS OF THE 10 EXEMPLIFICATIVE ANTIBODIES OBTAINED IN THE PRESENT INVENTION
Antibody name VH VL HC LC ADI-26624 44 54 74/88 75 ADI-29336 45 54 76/89 75 ADI-29340 46 54 77/90 75
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Antibody name VH VL HC LC ADI-26630 47 55 78/91 79 ADI-29341 48 55 80/92 79 ADI-29349 49 55 81/93 79 ADI-26591 50 57 82/94 83 ADI-29371 51 57 84/95 83 ADI-30793 52 58 85/96 86 ADI-30794 53 58 87/97 86
[00265] The following control antibodies used in the Example were expressed in 293HEK cells and purified:
[00266] Hu5F9 is an anti-human CD47 antibody, transiently expressed in 293 HEK cells, with the same sequence as that of antibody 5F9 in US Patent 2015/0183874 A1. AB6.12 is a humanized anti-CD47 antibody, transiently expressed in 293 HEK cells, with the same sequence as that of the AB6.12 antibody in US Patent 9045541. The AB6.12 antibody described in US Patent 9045541 is an anti-CD47 antibody that will not result in apparent cell agglutination.
[00267] For the transient expression of the antibody in 293HEK cells, a pTT5 vector was used. First, the heavy and light chains of the antibody were cloned into the single pTT5 vector. The vector pTT5 carrying the heavy and light chains of the antibody was transfected with chemical transfection in 293HEK cells. The chemical transfection reagent used is PEI (purchased from Polysciences) and the
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84/99 293HEK cells transiently transfected were performed according to the protocol provided by the manufacturer. First, plasmid DNA and transfection reagents were prepared on a clean bench, each half of the F17 culture medium (Gibco) (whose volume is 1/5 of the transfection volume) was added to a 50 ml centrifuge tube, with one half being supplemented with the filtered plasmid (130 pg / 100 ml) and the other half being supplemented with the filtered PEI (1 g / L, Polysciences) (the mass ratio (plasmid: PEI) = 1: 3), each well mixed for 5 min. Then, the two halves were carefully mixed 20 times and left to stand for 15-30 min, with no more than 30 min allowed. The DNA / PEI mixture was poured gently into 293HEK cells and mixed well. The cells were grown under 37 ° C, 8% CO2 for 7 days and fresh medium was added every 48 hours. After 7 days or continuous culture until cell viability <60%, a centrifugation was performed at 13000 rpm for 20 min. The supernatant was collected and purified on the Protein A column with the antibody purity> 95%.
EXAMPLE 2: DETERMINATION OF THE AFFINITY OF AN ANTI-CD47 ANTIBODY OF THE INVENTION [00268] The equilibrium dissociation constant (kD) of the 10 exemplary antibodies mentioned above in the present invention for human CD47 (hCD47) (Fab fragments used in the monovalence assay for to discard the impact potential by Fc fragments) was determined with the light biointerferometry assay (ForteBio).
[00269] The ForteBio affinity assay was performed in general as previously described (Estep, P., et a /., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5 (2): pages 270-8). In summary, the sensors were balanced off-line in assay buffer for 30 min and then
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85/99 tested online for 60 seconds to establish the baseline. The purified antibody obtained as described above was placed online over the AHQ sensor (ForteBio) to perform the ForteBio affinity measurement. Then, the sensor loaded with the antibody was exposed to 100 nM of the CD47 antigen for 5 min, followed by transferring the sensor to the assay buffer for 5 min to measure the dissociation rate. A kinetic analysis was performed using the 1: 1 connection model.
[00270] In the test performed as described in said assay, the affinities of ADI-26624, ADI-26630, ADi-26591, ADI-29336, ADI29340, ADI-29341, ADI-29349, ADI-29371, ADI-30793 and ADI -30794 are shown in Table 2.
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TABLE 2: BINDING THE ANTIBODY OF THE INVENTION IN IGG1 FORMAT, MEASURED BY LIGHT BIOINTERFEROMETRY
Antibody ForteBio Image: Human CD47-Fc at the tip of the AHQ, the antibody in Fab format in a solution (100 nM) [monovalent] ForteBio Image: The antibody in lgG1 format at the tip of the AHQ, human CD47-Fc in a solution (100 nM) [bivalent] ForteBio Image: The lgG1 antibody at the tip of the AHQ, cynomolgus CD47-Fc in a solution (100 nM) [bivalent] ForteBio Image: The antibody in lgG1 format at the tip of the AHQ, mouse CD47-Fc in a solution (100 nM) [bivalent] ADi-26591 N.B. 5.38E-09 3.39E-09 N.B. ADI-29371 2.18E-07 1.95E-09 1.41E-09 N, B, ADI-30793 9.34E-09 6.32E-10 5.84E-10 4.57E-09 ADI-30794 3.13E-09 5.62E-10 7.84E-10 N.B, ADI-26624 6.578E-08 1.054E-09 7.827E-10 N, B, ADI-29336 1.325E-09 4,768E-10 5.43626E-10 N, B, ADI-29340 5.484E-09 4.885E-10 6.12182E-10 2.12E-08 ADI-26630 3.567E-08 8.37E-10 7.27E-10 N, B, ADI-29341 4.623E-09 5.Q06E-10 5.30363E-10 N, B, ADI-29349 4.511E-09 5.552E-10 5.67054E-10 1.72E-08 Hu5F9 1.66E-08 4.20E-10 6.41E-10 1,266E-08
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Note: N.B. indicates no connection.
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It can be seen that all 10 exemplary antibodies mentioned above in the invention have very high affinities, comparable to the affinity of Hu5F9, an anti-CD47 antibody known and recognized in the art.
EXAMPLE 3: THE ANTI-CD47 ANTIBODY OF THE PRESENT INVENTION BINDING HUMAN CD47 [00272] In an assay based on flow cytometry, the binding of the 10 exemplary antibodies mentioned above in the invention to human CD47 was measured.
[00273] CHO cells that overexpress human CD47 (CHO-hCD47 cells) were created by transfecting CHO cells with a pCHOl.O (Invitrogen) vector that carries a cloned human CD47 cDNA (Biological Bell) at the multiple cloning site (MCS). CHO ~ hCD47 cells (0.2><10 ⁶ cells) were mixed with the test antibodies at different concentrations (the 10 exemplary antibodies mentioned above in the invention and Hu5F9, the maximum concentration of 900 nM, triple dilution, fully tested in 11 concentrations) in PBS with 0.1% bovine serum albumin (BSA) and incubated on ice for 30 min. Then, the cells were washed at least twice and incubated with a secondary antibody (PE-labeled goat anti-human IgG antibody, Southern Biotech, final concentration of 5 pg / ml) in PBS with 0.1% BSA in ice for 30 min (in the dark). The cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed using the Accuri C6 System (BD Biosciences) and a concentration-dependent graph was equipped with GraphPad, according to the cells' MFI.
[00274] ADI-26591, ADI-26624 and ADI-26630 (in lgG1 format, expressed in yeast) bind to hCD47 overexpressed in CHO cells (SEQ ID NO: 56) with EC50 values of 3.77 nM; 2.254 nM and
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3,895 nM, respectively, compatible with the ability to bind the control antibody Hu5F9 to hCD47 overexpressed in CHO cells (p EC50 value of the Hu5F9 control antibody being 3.726 nM) (see Figure 1).
[00275] In the test performed as described in the test above, ADI29336, ADI-29340, ADI-29341, ADI-29349, ADI-29371, ADI-30793 and ADI-30794 in lgG1 format produced in yeast cells bind to hCD47 overexpressed in CHO cells with EC50 values of 6.725 nM; 3.529 nM; 3,344 nM; 3.13 nM; 2,132 nM; 2,921 nM and 3,697 nM, respectively, essentially consistent with the ability to bind control Hu5F9 to CD47 overexpressed in CHO cells (with the EC50 value of 3.726 nM).
[00276] ADI-29336, ADI-29340, ADI-29341, ADI29349 and ADI-29371 antibodies in IgG4 format produced in CHO cells bind to overexpressed hCD47 in CHO cells with EC50 values of 2.475 nM; 2.194 nM; 1,892 nM; 2,043 nM and 2.31 nM, respectively, the affinities of these antibodies to hCD47 at the cellular level being, on the whole, greater than that of the Hu5F9 control antibody (with the EC50 value of 3.726).
EXAMPLE 4. THE ANTI-CD47 ANTIBODY OF THE INVENTION THAT BLOCKS THE INTERACTION OF THE BINDING OF HUMAN SIRPA CD47 WITH ACD47 [00277] The ability of the 10 exemplary antibodies to block the binding of human CD47 to SIRPa was measured with flow cytometry.
[00278] 0.2x10 ⁶ of the CHO cells expressing human CD47 prepared as described previously in Example 3 were matched with the test antibodies (ADI-26624, ADI-26630, ADI-29336, ADI-29340, ADI-29341, ADI-29349 and Hu5F9, the maximum concentration of 900 nM, triple dilution, fully tested in 11 concentrations) and
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200 nM of an Fc-labeled SIRPa protein (Acro Biosystems) in PBS with 0.1% BSA on ice for 30 min. Then, the cells were washed 3 times and subsequently incubated with a secondary antibody, goat anti-mouse IgG-APC (Allophycocyanin) (Biolegend), in PBS with 0.1% BSA on ice for 30 min (in the dark) . The cells were washed 3 times. The flow cytometry assay was performed on Accuri C6 System (BD Biosciences) and the MFI was calculated using the C6 software.
[00279] The capacities of ADI-26624, ADI-29336, ADI-29340, ADI-29371, ADI-26630, ADI-29341 and ADI-29349 in lgG1 format produced in yeast cells to block the binding of human SIRPaAPC to CD47 are consistent with that of the control antibody AB6.12.
[00280] Specifically, the IC50 values for the abilities of ADI-26624, ADI-29336 and ADI-29340 to block the binding of human SIRPaAPC to CD47 are 11.2 nM; 8.548 nM and 5.081 nM, respectively. The IC50 values for the capabilities of ADI-26630, ADI29341 and ADI-29349 to block the binding of human SIRPa-APC to CD47 are 2,986 nM; 2,476 nM and 3,097 nM, respectively. The IC50 value for the ability of the control antibody AB6.12 to block the binding of human SIRPa-APC to CD47 is 3,385 nM. (See Figure 2).
[00281] The capacities of ADI-26624, ADI-26630, ADI-29336, ADI-29340, ADI-29341 and ADI-29349 in lgG4 format produced in CHO cells to block the binding of human SIRPa-APC to CD47 are slightly higher than those of the control antibodies AB6.12 and Hu5F9.
[00282] Specifically, the IC50 values for the capabilities of ADI-26624, ADI-29336 and ADI-29340 to block the binding of human SIRPaAPC to CD47 are 1.043 nM; 1,389 nM and 1,223 nM, respec
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90/99 tively. IC50 values for the capabilities of ADI-26630, ADI29341 and ADI-29349 to block the binding of human SIRPa-APC to CD47 are 1.123 nM; 0.6042 nM and 0.7355 nM, respectively. The IC50 values for the ability of the control antibodies AB6.12 and Hu5F9 to block the binding of human SIRPa-APC to CD47 are 1.768 nM and 1.843 nM, respectively. (See Figure 3).
EXAMPLE 5. CAPACITY DETECTION OF AN ANTI-CD47 ANTIBODY OF THE INVENTION TO FACILITATE MACROPHAGIC TUMOR CELL Phagocytosis [00283] In an assay based on flow cytometry, the capabilities of the antibodies of the invention were measured (ADI-26624, ADI29336 , ADI-29340, ADI-26630, ADI-29341, ADI-29349, ADI-29371, ADI-30793 and ADI-30794) of facilitating tumor cell phagocytosis by macrophages were measured.
[00284] Fresh blood taken from a donor was subjected to density gradient centrifugation, resulting in peripheral blood mononucleated cells (PBMCs). CD14 positive monocytes were obtained and purified from isolated PBMCs according to the instructions of a kit (EasySep ™ Human CD14 Positive Selection Kit, Steam Cell) and 10 ng / mL granulocyte-macrophage colony stimulating factor (GM-CSF , R&D Systems) were added, followed by adherent culture for 7 consecutive days, during which 20 ng / mL of interferon-γ (IFN-γ, Acro Biosystem) was added for 1 hour stimulation on Day 5 and then 100 ng / mL of lipopolysaccharide (LPS, Sigma) were added for additional 48 hour stimulation. Thus, monocytes were induced in macrophages. The target tumor cells CCRF-CEM (acquired from ATCC) were fluorescently labeled according to the instructions of the CFSE CelITraceTM kit. The labeled tumor cells were co-cultured with the differentiated macrophage mentioned above at a
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91/99 4: 1 ratio, while the test antibodies were added in different concentrations and incubated at 37 ° C for 3 hours. Then, the cells were washed at least twice, followed by the addition of an allophicocyanin-labeled anti-CD14 antibody (APC) (purchased from BD), and incubated in PBS with 0.1% BSA on ice for 30 min ( in the dark). The cells were washed at least twice and analyzed by flow cytometry. The population of phagocytized cells is that of living cells that are positive for both CD14 and fluorescent dye CFSE (carboxyfluorescein diacetate, succinimidyl ester).
[00285] ADI-29336, ADI-29340, ADI-29341, ADI-29349, ADI-30793 and ADI-30794 in lgG1 format produced in yeast cells all have a great capacity to facilitate phagocytosis of tumor cells by macrophages . The ability of ADI-29340 to facilitate phagocytosis of tumor cells by macrophages is greater than that of control antibodies Hu5F9 and AB6.12, although the ability of ADI30793 and ADI-30794 to facilitate phagocytosis of tumor cells by macrophages is comparable that of the control antibody AB6.12 (see Figures 4 and 5.
[00286] ADI-26624 and ADI-26630 in lgG4 format produced in CHO cells can effectively facilitate phagocytosis of tumor cells by macrophages. The capabilities of ADI-26624 and ADI-26630 to facilitate phagocytosis of tumor cells by macrophages are consistent with that of the control antibody Hu5F9 (see Figure 6).
[00287] ADI-29336, ADI-29340, ADI-29341, ADI-29349 and ADI29371 in lgG4 format produced in CHO cells have, all, great capacity to facilitate the phagocytosis of tumor cells by macrophages. It can be seen from the results that the capabilities of ADI-29336, ADI-29340, ADI-29341 and ADI-29349 to significantly facilitate tumor cell phagocytosis by macrophages are significantly
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92/99 greater than the ability of the Hu5F9 control antibody, and the ability of ADI-29371 to facilitate phagocytosis of tumor cells by macrophages is comparable to that of the Hu5F9 control antibody (see Figure 7).
EXAMPLE 6. ANTITUMORAL ACTIVITY OF AN ANTICD47 ANTIBODY OF THE INVENTION [00288] The anti-tumor efficacy of the anti-CD47 antibody of the invention26624 (ADI-26624, ADI-26630, ADI-29340 and ADI29341) is studied in a NOD / SCID mouse model.
[00289] The procedure is as follows:
[00290] Human Raji cells from Burkitt's lymphomas (ATCC # CCL86) were purchased from ATCC and routinely subcultured strictly in accordance with ATCC requirements for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 107 cells / ml. 0.1 ml of the cell suspension was extracted and mixed with Matrigel 1: 1 to be inoculated subcutaneously in the right flank of NOD / SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). The tumor and body weight were measured twice a week throughout the study. The mice were euthanized when the tumor outcomes were achieved or the mice had> 20% body weight loss. 10 days after inoculation, the mice eligible for the experiment were randomized with 8 animals per group. Tumor volumes in mice were measured by a caliper with the following formula: (width) 2 x length / 2 in each group, with the maximum tumor volume in each group of mice being 110 mm3.
[00291] First, depositors studied the effectiveness of the ADI-26624 and ADI-26630 antibodies of the present invention to inhibit the tumor.
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93/99 [00292] The mice obtained by the method mentioned above were randomized and subjected to different treatments: intraperitoneal injection of 1 mg / kg or 5 mg / kg of PBS, the control IgG antibody (lgG4), Benchmark (Hu5F9) and the antibodies ADI-26624 and ADI-26630 of the present invention with a frequency of administration once every two days for 2 consecutive weeks. The detailed grouping and the mode of administration are shown in Table 3:
TABLE 3
Group Inoculated Cells Treatment modality1 Raji cells: Matrigel (1: 1) PBS2 Raji cells: Matrigel (1: 1) IgG control (5 mg / kg) once every two days for two consecutive weeks 3 Raji cells: Matrigel (1: 1) Hu5F9 (5 mg / kg) once every two days for two consecutive weeks 4 Raji cells: Matrigel (1: 1) Hu5F9 (1 mg / kg) once every two days for two consecutive weeks 5 Raji cells: Matrigel (1: 1) AD126624 (5 mg / kg) once every two days for two consecutive weeks 6 Raji cells: Matrigel (1: 1) AD126624 (1 mg / kg) once every two days for two consecutive weeks 7 Raji cells: Matrigel (1: 1) AD126630 (5 mg / kg) once every two days for two consecutive weeks 8 Raji cells: Matrigel (1: 1) AD126630 (1 mg / kg) once every two days for two consecutive weeks
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94/99 [00293] At the end of the experiment, the tumor growth inhibition rate was calculated with the following formula:
TGI% ”100% ^x ((TVOlpos-treatment with PBS ~ Tvolpos-treatment with antibody) / (TVOlpos-treatment with PBS ~ Tvolantes of treatment with PBs)), where Tvolpos-treatment with pbs is the tumor volume after completion of the experiment in the PBS Group of null control, Tvol post-treatment with antibody is the tumor volume after the conclusion of the experiment in the groups of antibodies (IgG, Hu5F9 and the antibodies of the present invention), and Tvolantes of the treatment with pbs is the initial tumor volume in the PBS Group of null control. [00294] For the experimental result, see figures 8, 9 and Table 4 below. It can be seen that the anti-CD47 monoclonal antibodies ADI-26624 and ADI-26630 in lgG4 format of the present patent application expressed in CHO cells significantly inhibit tumor growth in relation to the control IgG (equitech-Bio) and the control antibody Hu5F9.
[00295] The rates of tumor growth inhibition in Branches ADI-26630 of 1 mg / kg, ADI-26630 of 5 mg / kg, ADI-26624 of 1 mg / kg and ADI-26624 of 5 mg / kg are 100%, 104%, 79%, 94%, respectively. The tumors disappeared completely in 5/8 mice in the ADI-26630 branch of 5 mg / kg, and in 2/8 mice in the ADI-26630 branch of 1 mg / kg, in which the number of animals with complete disappearance of the tumor in both Branches are higher than in the Hu5F9 control antibody branch at the same dose (2 animals in Branches at 5 mg / kg and 1 animal in Branches at 5 mg / kg) (Table 4). The mice in all Branches in this study had no significant change in body weight 32 days after inoculation.
[00296] Thus, it can be seen that the antibody of the invention has a very good therapeutic effect on the tumor, which is superior to the therapeutic effect of the Hu5F9 control antibody.
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TABLE 4: STATISTICAL TABLE FOR TUMOR SIZE AND TUMOR GROWTH INHIBITION RATE IN THE STUDY WITH THE PRESENT ANTIBODY IN IgG4 FORMAT EXPRESSED IN CHO CELLS
Group Initial tumor volume (mm ³ ) Tumor volume at the end of the experiment (mm ³ ) Growth inhibition ratio (%) The number of animals with complete disappearance of tumors Proportion of animals with complete disappearance of tumors (%) PBS (null control) 111 17110/8 0 Human IgG (negative control), 5 mg / kg 110 1496 13 0/8 0 Hu5F9, 1 mg / kg 110 207 94 1/8 12 Hu5F9, 5 mg / kg 111 119 100 2/8 25 ADI-26630, 1 mg / kg 110 113 100 2/8 25 ADI-26630, 5 mg / kg 109 48 104 5/8 63 ADI-26624, 1 mg / kg 110 445 79 0/8 0 ADI-26624, 5 mg / kg 110 215 94 0/8 0
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96/99 [00297] Next, the present inventors proceed with the detection of inhibitory effects of antibodies ADP29340 and ADI29341 on the tumor.
[00298] The mice obtained by the method mentioned above were randomized and subjected to different treatments, that is, the intraperitoneal injection of 0.5 mg / kg or 5 mg / kg of PBS, the control IgG antibody (lgG4) and the antibodies ADI-26630, ADI-29340 and ADI29341 of the present invention, once every two days for 2 consecutive weeks. The detailed grouping and mode of administration are shown in Table 5 below:
Table 5
Group Inoculated Cells Treatment modality1 Raji cells:Matrigel (1: 1) PBS2 Raji cells:Matrigel (1: 1) Control of human IgG (5 mg / kg) once every two days for two consecutive weeks 3 Raji cells:Matrigel (1: 1) AD126630 (5 mg / kg). once every two days for two consecutive weeks 4 Raji cells:Matrigel (1: 1) AD126630 (0.5 mg / kg). once every two days for two consecutive weeks 5 Raji cells:Matrigel (1: 1) AD1269340 (5 mg / kg). once every two days for two consecutive weeks 6 Raji cells:Matrigel (1: 1) AD 129340 (0.5 mg / kg). once every two days for two consecutive weeks 7 Raji cells:Matrigel (1: 1) AD1269341 (5 mg / kg). once every two days for two consecutive weeks 8 Raji cells:Matrigel (1: 1) AD 129341 (0.5 mg / kg). once every two days for two consecutive weeks
[00299] At the end of the experiment, the rate of tumor growth inhibition was calculated with the formula mentioned above. The anti-CD47 monoclonal antibodies ADI-26630, ADI-29340 and
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ADI-29341 in lgG4 format of the present patent application expressed in CHO cells could significantly inhibit tumor growth (Table 6, Figure 10 and Figure 11).
[00300] The tumor growth inhibition rate in the ADI-26630 Branches of 0.5 mg / kg, ADI-26630 of 5 mg / kg, ADI-29340 of 0.5 mg / kg, ADI-29340 of 5 mg / kg, ADI-29341 of 0.5 mg / kg and ADI29341 of 5 mg / kg are 99%, 110%, 103%, 109%, 104% and 109%, respectively. The tumors disappeared completely in 5/8 mice in the 0.5 mg / kg ADI-29340 branches, 4/8 mice in the 0.5 mg / kg ADI-29341 branch and in 8/8 mice in the ADI-26630 branches and ADI-29340 of 5 mg / kg (Table 6). The mice in all Branches in this study had no significant change in body weight 30 days after inoculation.
Thus, it can be seen that the antibody of the invention has a very good therapeutic effect on the tumor.
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TABLE 6: STATISTICAL TABLE FOR TUMOR SIZE AND TUMOR GROWTH INHIBITION RATE IN THE STUDY WITH THE PRESENT ANTIBODY IN IGG4 FORMAT EXPRESSED IN CHO CELLS
Group Initial tumor volume (mm ³ ) Tumor volume at the end of the experiment (mm ³ ) Growth inhibition ratio (%) The number of animals with complete disappearance of tumors Proportion of animals with complete disappearance of tumors (%) PBS (null control) 101 10470/8 0 h-lgG, 5 mg / kg 100 10890/8 0 ADI26630, 0.5 mg / kg 99 113 99 1/8 12.5 AD 126630, 5 mg / kg 99 0 110 8/8 100 ADI29340, 0.5 mg / kg 98 69 103 5/8 62.5 ADI29340, 5 mg / kg 99 8 109 8/8 100 ADI29341.0.5 mg / kg 99 60 104 4/8 50 ADI29341.5 mg / kg 99 12 109 7/8 87.5
EXAMPLE 7. DETECTION OF THE ACTIVITY OF AN ANTI-CD47 ANTIBODY OF THE INVENTION TO FACILITATE RBC AGGLUTINATION.
98/99 [00302] It is known in the art that most anti-CD47 antibodies have the side effect of facilitating the agglutination of RBC, thus limiting the therapeutic applications of these antibodies. Therefore, the present inventors have further investigated the RBC agglutination of the antibodies described in the present patent application.
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99/99 [00303] The test procedure is as follows:
[00304] Fresh human blood is collected and washed three times with PBS to prepare a 10% suspension of human RBC. Human RBCs are incubated with the test antibody (maximum concentration of 60 µg / ml, triple dilution, total of 11 concentrations) in a 96-well round bottom plate at 37 ° C for 2-6 hours. After completion of the reaction, a photograph is taken and the result is evaluated. The criteria for judging the result is that the RBCs agglutination reaction occurs if the red blood cells settle and spread in cross-linking at the bottom of the well, appearing as a haze (see the result for Hu5F9 in Figure 12), and the agglutination reaction of RBCs does not occur if the RBCs settle on a dotted red dot at the bottom of the well (see the control in Figure 12).
[00305] In the test performed as described in the above test, the result of the hemagglutination reaction was shown in Figure 12. It can be seen in Figure 12 that the activity of ADI26630, ADI29340 and ADI29341 in RBC agglutination is very weak, and that its activity to facilitate agglutination of RBC is significantly less than that of the Hu5F9 control, and comparable to that of the AB6.12 control. It can be seen that the antibodies described in the present patent application significantly decreased the agglutination of blood cells and, consequently, can result in the significantly reduced side effect in the clinical treatment model, and can be widely used in the treatment of various cancers.

权利要求:
Claims (23)
[1]
1. Isolated anti-CD47 monoclonal antibody or a fragment that binds to its antigen, characterized by the fact that it comprises:
(i) three regions determining the HCDRs complementarity of the heavy chain variable region, as shown in SEQ ID NO: 44, 45 or 46, and three regions determining the LCDRs complementarity of the variable region of a light chain, as shown in SEQ ID NO : 54;
(ii) three regions determining the HCDRs complementarity of the variable region of the heavy chain, as shown in SEQ ID NO: 47, 48 or 49, and three regions determining the complementarity LCDRs of the variable region of a light chain, as shown in SEQ ID NO. : 55;
(iii) three regions determining the HCDRs complementarity of the variable region of the heavy chain, as shown in SEQ ID NO: 50 or 51, and three regions determining the LCDRs complementarity of the variable region of a light chain, as shown in SEQ ID NO: 57 ; or (iv) three HCDRs complementarity determining regions of the heavy chain variable region, as shown in SEQ ID NO: 52 or 53, and three LCDRs complementarity determining regions of the light chain variable region, as shown in SEQ ID NO: 58.
[2]
2. Isolated anti-CD47 monoclonal antibody or an antigen binding fragment thereof, characterized by the fact that it comprises three HCDRs complementarity determining regions of the heavy chain variable region and three LCDRs complementarity determining regions of the light chain variable region, where HCDR1 comprises the amino acid sequence containing
Petition 870190067389, of 7/17/2019, p. 204/216
2/8 as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 or 99, HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 or 101, HCDRS comprises the amino acid sequence as shown in SEQ ID NO: 17, 18, 19, 20, 21, 22, 102 or 103, LCDR1 comprises the sequence of amino acids as shown in SEQ ID NO: 23 or 24, LCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 25 or 26, and LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 27; 28; 29 or 30.
[3]
3. Isolated anti-CD47 monoclonal antibody or an antigen binding fragment thereof, characterized by the fact that it comprises a variable region of the heavy chain and / or a variable region of the light chain, in which the variable region of the heavy chain comprises:
(i) three complementarity determining regions (HCDRs) contained in the VH of any of the antibodies listed in table B;
(ii) the combination of HCDR1, HCDR2 and HCDR3 shown in Table A;
(iii) HCDR1, HCDR2 and HCDR3, where HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 98 or 99, HCDR2 comprises the sequence of amino acids shown in SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 100 or 101, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20, 21 , 22, 102 or 103; or (iv) the variant of HCDRs in (i) to (iii), comprising a substitution (for example, a conservative substitution), elimination or insertion of amino acids of at least one amino acid and no more than 5 amino acids in the three CDR regions ; and / or the variable region of the light chain comprises:
Petition 870190067389, of 7/17/2019, p. 205/216
3/8 (i) three complementarity determining regions (LCDRs) contained in the VL of any of the antibodies listed in Table B;
(ii) the combination of LCDR1, LCDR2 and LCDR3 shown in Table A;
(iii) LCDR1, LCDR2 and LCDR3, where LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23 or 24, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25 or 26, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; 28; 29 or 30; or (iv) the LCDR variant in (i) to (iii), comprising a substitution (for example, a conservative substitution), elimination or insertion of amino acids of at least one amino acid and no more than 5 amino acids in the three CDR regions ;
[4]
4. Isolated monoclonal antibody or an antigen-binding fragment thereof, according to any one of claims 1 to 3, characterized by the fact that it comprises:
(i) a heavy chain variable region comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 44, 45 or 46, and / or a light chain variable region comprising a amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 54, or (ii) a heavy chain variable region comprising an amino acid sequence with at least 90% sequence identity to the sequence of amino acids shown in SEQ ID NO: 47, 48 or 49, and / or a variable region of the light chain comprising an amino acid sequence with at least 90% of the sequence identity to the amino acid sequence shown in SEQ ID NO:
Petition 870190067389, of 7/17/2019, p. 206/216
4/8
55, or (iii) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 50 or 51, and / or a light chain variable region comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:
57, or (iv) a heavy chain variable region comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 52 or 53, and / or a light chain variable region comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:
58.
[5]
5. Isolated monoclonal antibody or an antigen-binding fragment thereof, according to any one of claims 1 to 4, characterized by the fact that it comprises:
(i) a heavy chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 74, 76, 77, 88, 89 or 90, and / or a light chain comprising a amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 75, or (ii) a heavy chain comprising an amino acid sequence with at least 90% sequence identity to the shown amino acid sequence in SEQ ID NO: 78, 80, 81, 91, 92 or 93, and / or a light chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 79, or
Petition 870190067389, of 7/17/2019, p. 207/216
5/8 (iii) a heavy chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 82, 84, 94, or 95, and / or a light chain comprising a amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 83, or (iv) a heavy chain comprising an amino acid sequence with at least 90% sequence identity to the shown amino acid sequence in SEQ ID NO: 85, 87, 96, or 97, and / or a light chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 86.
[6]
6. Isolated monoclonal antibody or an antigen-binding fragment thereof, according to any one of claims 1 to 5, characterized in that said antibody is a humanized antibody or human antibody.
[7]
7. Isolated monoclonal antibody or an antigen-binding fragment thereof, according to any of claims 1 to 6, characterized in that said antigen-binding fragment is selected from the group consisting of Fab fragment, Fab'-SH, Fv, scFv or (Fab ') 2.
[8]
An isolated monoclonal antibody or an antigen-binding fragment thereof, according to any one of claims 1 to 7, comprising a structural sequence, characterized by the fact that at least a part of the structural sequence is a human consensus structural sequence.
[9]
9. Anti-CD47 monoclonal antibody or an antigen-binding fragment thereof, characterized by the fact that it competes with the antibody as defined in any one of claims 1 to 8, for binding to CD47, or antagonizes and / or blocks the binding of the
Petition 870190067389, of 7/17/2019, p. 208/216
6/8
CD47 to the antibody as defined in any one of claims 1 to 8.
[10]
10. Isolated nucleic acid, characterized by the fact that it encodes isolated anti-CD47 monoclonal antibodies or antigen-binding fragments thereof as defined in any one of claims 1 to 9.
[11]
11. Vector characterized by the fact that it comprises the nucleic acid as defined in claim 10, said vector being preferably an expression vector.
[12]
12. Host cell characterized by the fact that it comprises the vector as defined in claim 11, said host cell being preferably a prokaryotic or eukaryotic cell; more preferably being selected from the group consisting of yeast cells, mammalian cells or other cells that are suitable for preparing antibodies or antigen-binding fragments, and preferably being CHO cells and 293 cells.
[13]
13. Method for preparing the anti-CD47 monoclonal antibody or an antigen-binding fragment thereof, characterized by the fact that it comprises culturing the host cell as defined in claim 12, under a condition that is suitable for expressing the nucleic acid encoding the monoclonal anti-CD47 antibodies or antigen-binding fragments thereof as defined in any one of claims 1 to 9, optionally isolating the monoclonal antibodies or antigen-binding fragments thereof, and, optionally, the method further comprises recovering the monoclonal anti-CD47 antibodies or antigen-binding fragments thereof from the host cell.
[14]
14. Anti-CD47 monoclonal antibodies or antigen-binding fragments thereof, characterized by the fact that they are
Petition 870190067389, of 7/17/2019, p. 209/216
7/8 prepared by the method as defined in claim 13.
[15]
15. Pharmaceutical composition, characterized by the fact that it comprises anti-CD47 antibodies or antigen-binding fragments thereof as defined in any one of claims 1 to 9 and 14, and optionally pharmaceutical vehicles.
[16]
16. Method for facilitating phagocytosis by macrophages in an individual, characterized in that it comprises administering to the individual the effective amount of anti-CD47 antibodies or antigen-binding fragments thereof as defined in any of claims 1 to 9 and 14 , or the effective amount of the pharmaceutical composition as defined in claim 15.
[17]
17. Method for treating a cancer or tumor in an individual, characterized in that it comprises administering to the individual the effective amount of anti-CD47 antibodies or antigen-binding fragments thereof as defined in any one of claims 1 to 9 and 14, or the effective amount of the pharmaceutical composition as defined in claim 15.
[18]
18. Method for relieving the symptoms of a cancer or tumor, characterized in that it comprises administering to an individual in need the effective amount of anti-CD47 antibodies or antigen-binding fragments thereof as defined in any one of claims 1 to 9 and 14, or the effective amount of the pharmaceutical composition as defined in claim 15.
[19]
19. Method according to any of claims 16 to 18, characterized by the fact that the individual is a human being.
[20]
20. Method according to claim 17 or 18, characterized by the fact that the cancer or tumor is several hematological neoplasms and solid tumors, for example, acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), lymphocytic leukemia
Petition 870190067389, of 7/17/2019, p. 210/216
Acute 8/8 (ALL), chronic lymphocytic leukemia (CLL) non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), lymphoma, breast carcinoma, head and neck cancer, gastric carcinoma, lung cancer, esophageal carcinoma, carcinoma intestinal, ovarian carcinoma, cervical carcinoma, liver carcinoma, renal carcinoma, pancreatic carcinoma, bladder carcinoma, colorectal cancer, glioma, melanoma and other solid tumors.
[21]
21. Method according to any one of claims 16 to 18, characterized in that it also comprises administering to the individual an effective amount of one or more other medications, the other medications, for example, being several medications with monoclonal antibody that attack tumor cells through T cell recognition, for example, rituximab, cetuximab and trastuzumab.
[22]
22. Method for detecting the presence of the CD47 protein in a sample, characterized by the fact that it comprises:
(a) placing the samples in contact with the antibody or antigen-binding fragment thereof as defined in any of claims 1 to 9 and 14; and (b) detecting the formation of the complex between the antibody or antigen-binding fragment thereof and the CD47 protein.
[23]
23. Method for determining the effectiveness of a tumor therapy, characterized by the fact that it comprises determining the number of cancer cells that express CD47 in a sample from an individual before and after therapy, in which the reduced number of cancer cells that express CD47 indicates that the therapy is effective.

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

优先权:

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申请号 | 申请日 | 专利标题

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CN201710759828.9A|CN109422811A|2017-08-29|2017-08-29|Anti-cd 47 antibody and application thereof|

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