Manufacture of antibodies for tumor-selective binding to CD47

Abstract

An antibody is provided comprising at least one Fab portion that binds to CD47 and at least one Fab portion that binds to the tumor-associated antigen (TAA) CD20; wherein the Fab portion that binds to CD47 exhibits low affinity for CD47; wherein the Fab portion that binds to CD20 exhibits high affinity for CD20; wherein the antibody selectively binds to CD47 in tumor cells and blocks CD47 interaction with SIRPα but does not exhibit substantial binding to CD47 in normal cells.

Description

Technology Field

[0001] The present invention provides a tumor-selective antibody, a pharmaceutical composition, and a method of use for the treatment of pathological conditions affected by cells undergoing abnormal proliferation, including hematological oncological conditions, hematological malignancies, lymphoproliferative disorders, B-cell disorders, B-cell malignancies, and B-cell lymphoma.

[0002] Cross-reference regarding related applications

[0003] This application has priority under 35 USC §119(e) to U.S. Provisional Application No. 62 / 830,335 filed April 5, 2019, the full text of which is incorporated herein by reference.

[0004] Sequence list

[0005] The present application contains a list of sequences submitted in ASCII format via EFS-Web, the entirety of which is incorporated herein by reference. The filename of the ASCII copy created on March 26, 2020 is 298068-314_Sequence_Listing.txt and the size is 407 kilobytes. Background Technology

[0006] Over the past decade, the use of inhibitors against suppressive immune checkpoints has been one of the most significant developments in cancer therapy (Sharpe AH. Introduction to checkpoint inhibitors and cancer immunotherapy. Immunol Rev. 2017 Mar;276(1):5-8). The interesting results obtained from CTLA-4 and PD-1 blockades have led to the evaluation of several innate immune checkpoints that can be targeted in cancer therapy, particularly in pathways that regulate macrophage function. Macrophages express SIRPα, which interacts with CD47, a universally expressed protein that mediates the "don't eat me" signaling function that inhibits phagocytosis. The expression of CD47 confers resistance to the phagocytosis of antibody-bound tumor cells by macrophages. In the absence of CD47 binding to SIRPα, antibodies capable of binding to Fc-receptors on macrophages can enhance the phagocytosis of these cells. Cancer cells have evolved to dominate these interactions by upregulating the expression of CD47 on their cell surfaces, thereby balancing phagocytic signals and increasing the chance to evade innate immune surveillance (Matlung HL, Szilagyi K, Barclay NA, van den Berg TK. The CD47-SIRPα signaling axis as an innate immune checkpoint in cancer. Immunol Rev. 2017 Mar;276(1):145-164). Therefore, blocking the CD47-SIRPα interaction represents a promising therapeutic strategy for activating the phagocytic elimination of tumor cells from the body.Several SIRPα-CD47 blockers, including humanized and fully human anti-CD47 antibodies, anti-SIRPα antibodies, soluble SIRPα dimers fused to the Fc portion of human IgG, high-affinity monomeric SIRPα without the Fc portion, and camelid-derived monomeric fragments (nanobodies) of anti-CD47 antibodies, have demonstrated efficacy in in vitro and preclinical studies against various types of human tumors (Veillette A, Chen A., SIRP-CD47 Immune Checkpoint Blockade in Anticancer Therapy. Trends in Immunology, 2018, 39(3):173-184). Some SIRPα-CD47 blockers, including CC-90002 (anti-CD47), Forty Seven's anti-CD47 (Hu5F9-G4), and Trillium's SIRPα-fusion Fc, were tested in Phase I and Phase II clinical trials, respectively (Veillette A, Tang Z. Signaling Regulatory Protein (SIRP)α-CD47 Blockade Joins the Ranks of Immune Checkpoint Inhibition. J Clin Oncol. 2019 Feb 27:JCO190012). In clinical practice, these approaches are limited by the need for combination therapy (e.g., rituximab), tissue sinks in targeting CD47 using high-affinity binders (i.e., the presence of non-tumor cells to which the therapeutic antibody binds, resulting in reduced bioavailability of the antibody to tumor cells), and hematological toxicities (anemia, neutropenia, and / or thrombocytopenia) observed in some clinical molecules. Importantly, while protein therapeutics are actually being explored to treat a number of diseases, biopharmaceutical entities can trigger an immune response involving the production of anti-entity antibodies that result in reduced efficacy and / or toxicity when administered to subjects.

[0007] The present invention relates to an antibody comprising at least one Fab portion that binds to CD47 with low affinity and at least one Fab portion that binds to CD20 with high affinity; wherein the bispecific antibody selectively binds to CD47 in tumor cells and has substantially no binding to CD47 in normal cells.

[0008] The Fab portion described herein that binds to CD47 with low affinity generally exhibits an affinity for CD47 of, for example, about 0.1 μM to about 25 μM when measured as Kd (dissociation constant) by surface plasmon resonance (SPR). The Fab portion described herein that binds to CD47 with low affinity exhibits an affinity for CD47 of about 0.25 μM to about 20 μM. A specific preferred embodiment exhibits an affinity for CD47 of about 0.4 μM to about 4.0 μM. A specific embodiment exhibits an affinity for CD47 of about 1 μM to about 3.0 μM. In some embodiments, for example, the Fab portion binding to CD47 exhibits an affinity for CD47 of about 0.1 μM to about 5.0 μM. In some embodiments, the Fab portion binding to CD47 is about 0.1 μM to about 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 3.1 μM, 3.2 μM, 3.3 μM, It shows the affinity for CD47 at 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4.0 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, or about 5.0 μM.

[0009] In some embodiments, the Fab portion binding to CD47 exhibits an affinity for CD47 of about 0.2 μM to about 4.0 μM. In further embodiments, the Fab portion binding to CD47 exhibits an affinity for CD47 of about 0.5 μM to about 3.5 μM. In further embodiments, the Fab portion binding to CD47 exhibits an affinity for CD47 of about 1.0 μM to about 3.0 μM.

[0010] The present invention further relates to an antibody comprising at least one Fab portion that binds to CD47 and at least one Fab portion that binds to CD20; wherein the Fab portion that binds to CD47 exhibits low affinity for CD47; wherein the Fab portion that binds to CD20 exhibits high affinity for CD20; wherein the bispecific antibody selectively binds to CD47 in tumor cells and substantially has no binding to CD47 in normal cells; and wherein the antibody activates antibody-dependent phagocytosis in tumor cells expressing CD20.

[0011] The present invention further relates to an antibody comprising at least one Fab portion that binds to CD47 and at least one Fab portion that binds to CD20; wherein the Fab portion that binds to CD47 exhibits low affinity for CD47; wherein the Fab portion that binds to CD20 exhibits high affinity for CD20; wherein the bispecific antibody selectively binds to CD47 in tumor cells and substantially has no binding to CD47 in normal cells; wherein the antibody mediates complement-dependent cytotoxicity (CDC) in tumor cells expressing CD20.

[0012] The present invention further relates to an antibody comprising at least one Fab portion that binds to CD47 and at least one Fab portion that binds to CD20; wherein the Fab portion that binds to CD47 exhibits low affinity for CD47; wherein the Fab portion that binds to CD20 exhibits high affinity for CD20; wherein the bispecific antibody selectively binds to CD47 in tumor cells and substantially has no binding to CD47 in normal cells; and wherein the antibody mediates antibody-dependent cytotoxicity (ADCC) in tumor cells expressing CD20.

[0013] The present invention relates to the monomeric element of the IgG1 1+1 heteromer described herein, comprising a specific light chain (LC) and heavy chain (HC) constant region that induces the production of the IgG1 1+1 heteromer form. In one embodiment, an anti-CD47 LC constant region that reduces LS mismatch during production comprises SEQ ID NO: 340. In another embodiment, an anti-CD47 HC constant region that ensures the formation of a heteromer of Fc during production comprises SEQ ID NO: 342. In another embodiment, an anti-CD20 LC constant region that reduces LS mismatch during production comprises SEQ ID NO: 344. In another embodiment, an anti-CD20 HC constant region that ensures the formation of a heteromer of Fc during production comprises SEQ ID NO: 346.

[0014] The present invention relates to an antibody in which the Fab portion further binding to CD47 is derived from the VL (SEQ No.: 325) and VH (SEQ No.: 326) regions of the anti-CD47 species CC-90002, wherein the binding affinity for CD47 is substantially weakened and the immunogenicity is substantially reduced.

[0015] The present invention further relates to a bispecific antibody in which the anti-CD47 VL is derived from SEQ ID NO: 325 and exhibits 1-7 amino acid substitutions for SEQ ID NO: 325; and the anti-CD47 VH is derived from SEQ ID NO: 326 and exhibits 1-11 amino acid substitutions for SEQ ID NO: 326, wherein the binding-affinity for CD47 is substantially weakened and the immunogenicity is substantially reduced.

[0016] The present invention relates to an anti-CD47 VL derived from SEQ ID NO: 325 and exhibiting 1 to 7 amino acid substitutions for SEQ ID NO: 325, wherein at least one amino acid substitution in the anti-CD47 VL (SEQ ID NO: 325) is selected from the group consisting of A10S, M11L, K24R, A51E, N52S, L54F, and S56D; The present invention relates to a bispecific antibody in which (substitutions in anti-CD47 VL are numbered based on the first amino acid of VL), anti-CD47 VH is derived from SEQ ID NO: 326 and exhibits 1 to 11 amino acid substitutions for SEQ ID NO: 326, wherein at least one amino acid substitution in anti-CD47 VH (SEQ ID NO: 326) is selected from the group consisting of T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L (substitutions in anti-CD47 VH are numbered based on the first amino acid of VH).

[0017] The present invention relates to a bispecific antibody in which a portion further binding to CD47 comprises the following: (i) a light chain variable region (VL) region selected from the group consisting of the following: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, Sequence No.: 71, Sequence No.: 73, Sequence No.: 75, Sequence No.: 77, Sequence No.: 79, Sequence No.: 81, Sequence No.: 83, Sequence No.: 85, Sequence No.: 87, Sequence No.: 89, Sequence No.: 91, Sequence No.: 93, Sequence No.: 95, Sequence No.: 97, Sequence No.: 99, Sequence No.: 101, Sequence No.: 103, Sequence No.: 105, Sequence No.: 107, Sequence No.: 109, Sequence No.: 111, Sequence No.: 113, Sequence No.: 115, Sequence No.: 117, Sequence No.: 119, Sequence No.: 121, Sequence No.: 123, Sequence No.: 125, Sequence No.: 127, Sequence No.: 129, Sequence No.: 131, Sequence No.: 133, Sequence No.: 135, Sequence No.: 137, Sequence No.: 139, Sequence No.: 141, Sequence No.: 143, Sequence No.: 145, Sequence No.: 147, Sequence No.: 149, Sequence No.: 151, Sequence No.: 153, Sequence No.: 155, Sequence No.: 157, Sequence No.: 159, Sequence No.: 161, Sequence No.: 163, Sequence No.: 165, Sequence No.: 167, Sequence No.: 169, Sequence No.: 171, Sequence No.: 173, Sequence No.: 175, Sequence No.: 177, Sequence No.: 179, Sequence No.: 181, Sequence No.: 183, Sequence No.: 185, Sequence No.: 187, Sequence No.: 189, Sequence No.: 191,Sequence No.: 193, Sequence No.: 195, Sequence No.: 197, Sequence No.: 199, Sequence No.: 201, Sequence No.: 203, Sequence No.: 205, Sequence No.: 207, Sequence No.: 209, Sequence No.: 211, Sequence No.: 213, Sequence No.: 215, Sequence No.: 217, Sequence No.: 219, Sequence No.: 221, Sequence No.: 223, Sequence No.: 225, Sequence No.: 227, Sequence No.: 229, Sequence No.: 231, Sequence No.: 233, Sequence No.: 235, Sequence No.: 237, Sequence No.: 239, Sequence No.: 241, Sequence No.: 243, Sequence No.: 245, Sequence No.: 247, Sequence No.: 249, Sequence No.: 251, Sequence No.: 253, Sequence No.: 255, Sequence No.: 257, Sequence No.: 259, Sequence No.: 261, Sequence No.: 263, Sequence No.: 265, Sequence No.: 267, Sequence No.: 269, Sequence No.: 271, Sequence No.: 273, Sequence No.: 275, Sequence No.: 277, Sequence No.: 279, Sequence No.: 281, Sequence No.: 283, Sequence No.: 285, Sequence No.: 287, Sequence No.: 289, Sequence No.: 291, Sequence No.: 293, Sequence No.: 295, Sequence No.: 297, Sequence No.: 299, Sequence No.: 301, Sequence No.: 303, Sequence No.: 305, Sequence No.: 307, Sequence No.: 309, Sequence No.: 311, Sequence No.: 313, Sequence No.: 315, Sequence No.: 317, Sequence No.: 319, and Sequence No.: 321; and,

[0018] (ii) Heavy chain variable region (VH) region selected from the group consisting of the following: Sequence No. 2, Sequence No. 4, Sequence No. 6, Sequence No. 8, Sequence No. 10, Sequence No. 12, Sequence No. 14, Sequence No. 16, Sequence No. 18, Sequence No. 20, Sequence No. 22, Sequence No. 24, Sequence No. 26, Sequence No. 28, Sequence No. 30, Sequence No. 32, Sequence No. 34, Sequence No. 36, Sequence No. 38, Sequence No. 40, Sequence No. 42, Sequence No. 44, Sequence No. 46, Sequence No. 48, Sequence No. 50, Sequence No. 52, Sequence No. 54, Sequence No. 56, Sequence No. 58, Sequence No. 60, Sequence No. 62, Sequence No. 64, Sequence No. 66, Sequence No. 68, Sequence No. 70, Sequence No. 72, Sequence No. 74, Sequence No. 76, Sequence No.: 78, Sequence No.: 80, Sequence No.: 82, Sequence No.: 84, Sequence No.: 86, Sequence No.: 88, Sequence No.: 90, Sequence No.: 92, Sequence No.: 94, Sequence No.: 96, Sequence No.: 98, Sequence No.: 100, Sequence No.: 102, Sequence No.: 104, Sequence No.: 106, Sequence No.: 108, Sequence No.: 110, Sequence No.: 112, Sequence No.: 114, Sequence No.: 116, Sequence No.: 118, Sequence No.: 120, Sequence No.: 122, Sequence No.: 124, Sequence No.: 126, Sequence No.: 128, Sequence No.: 130, Sequence No.: 132, Sequence No.: 134, Sequence No.: 136, Sequence No.: 138, Sequence No.: 140, Sequence No.: 142, Sequence No.: 144, Sequence No.: 146, Sequence No.: 148, Sequence No.: 150, Sequence No.: 152, Sequence No.: 154, Sequence No.: 156, Sequence No.: 158, Sequence No.: 160, Sequence No.: 162, Sequence No.: 164, Sequence No.: 166, Sequence No.: 168, Sequence No.: 170, Sequence No.: 172, Sequence No.: 174, Sequence No.: 176, Sequence No.: 178, Sequence No.: 180, Sequence No.: 182, Sequence No.: 184, Sequence No.: 186, Sequence No.: 188, Sequence No.: 190, Sequence No.: 192, Sequence No.: 194, Sequence No.: 196, Sequence No.: 198, Sequence No.: 200, Sequence No.: 202,Sequence No.: 204, Sequence No.: 206, Sequence No.: 208, Sequence No.: 210, Sequence No.: 212, Sequence No.: 214, Sequence No.: 216, Sequence No.: 218, Sequence No.: 220, Sequence No.: 222, Sequence No.: 224, Sequence No.: 226, Sequence No.: 228, Sequence No.: 230, Sequence No.: 232, Sequence No.: 234, Sequence No.: 236, Sequence No.: 238, Sequence No.: 240, Sequence No.: 242, Sequence No.: 244, Sequence No.: 246, Sequence No.: 248, Sequence No.: 250, Sequence No.: 252, Sequence No.: 254, Sequence No.: 255, Sequence No.: 258, Sequence No.: 260, Sequence No.: 262, Sequence No.: 264, Sequence No.: 266, Sequence No.: 268, Sequence No.: 270, Sequence No.: 272, Sequence No.:274, Sequence No.:276, Sequence No.:278, Sequence No.:280, Sequence No.:282, Sequence No.:284, Sequence No.:286, Sequence No.:288, Sequence No.:290, Sequence No.:292, Sequence No.:294, Sequence No.:296, Sequence No.:298, Sequence No.:300, Sequence No.:302, Sequence No.:304, Sequence No.:306, Sequence No.:308, Sequence No.:310, Sequence No.:312, Sequence No.:314, Sequence No.:316, Sequence No.:318, Sequence No.:320, and Sequence No.:322.,

[0019] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD20, comprising anti-CD20 VL CDR RASSSVSYIH (CDRL1; SEQ No.: 353), ATSNLAS (CDRL2; SEQ No.: 354), QQWTSNPPT (CDRL3; SEQ No.: 355); and VH CDR SYNMH (CDRH1; SEQ No.: 356), AIYPGNGDTSYNQKFKG (CDRH2; SEQ No.: 357), STYYGGDWYFNV (CDRH3; SEQ No.: 358).

[0020] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD20, comprising anti-CD20 LC (Sequence No.: 331) and anti-CD20 HC (Sequence No.: 332).

[0021] The present invention relates to a bispecific antibody comprising a light chain variable region (VL) region in which a Fab portion further binding to CD47 includes VL CDR QASQDIHRYLS (CDRL1; SEQ No.: 359), RESRFVD (CDRL2; SEQ No.: 360), and LQYDEFPYT (CDRL3; SEQ No.: 361); and a heavy chain variable region (VH) region in which VH CDR DYYLH (CDRH1; SEQ No.: 362), WIDPDQGDTYYAQKFQG (CDRH2; SEQ No.: 363), and AYGESSYPMDY (CDRH3; SEQ No.: 364).

[0022] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD47, a light chain variable region (VL) region comprising VL CDR RASQDIHRYLS (CDRL1; SEQ No.: 365), RESRFVD (CDRL2; SEQ No.: 366), and LQYDEFPYT (CDRL3; SEQ No.: 367); and a heavy chain variable region (VH) region comprising VH CDR DYYLH (CDRH1; SEQ No.: 368), WIDPDQGDTYYAQKFQG (CDRH2; SEQ No.: 369), and AYGESSYPMDY (CDRH3; SEQ No.: 370).

[0023] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD47, a light chain variable region (VL) region comprising VL CDR RASQDIHRYLS (CDRL1; SEQ No.: 371), RANRLVS (CDRL2; SEQ No.: 372), and LQYDEFPYT (CDRL3; SEQ No.: 373); and a heavy chain variable region (VH) region comprising VH CDR DYYLH (CDRH1; SEQ No.: 374), WIDPDQGDTYYAQKFQG (CDRH2; SEQ No.: 375), and AYGESSYPMDY (CDRH3; SEQ No.: 376).

[0024] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD47, VL (SEQ ID: 317); and VH (SEQ ID: 318).

[0025] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD47, comprising VL (SEQ ID: 319); and VH (SEQ ID: 320).

[0026] The present invention relates to a bispecific antibody comprising a Fab portion that further binds to CD47, VL (SEQ ID: 321); and VH (SEQ ID: 322).

[0027] The present invention relates to a bispecific antibody in which the Fab portion that further binds to CD47 comprises LC (Sequence No. 333) and HC (Sequence No. 334).

[0028] The present invention relates to a bispecific antibody in which the Fab portion that further binds to CD47 comprises LC (Sequence No. 335) and HC (Sequence No. 336).

[0029] The present invention relates to a bispecific antibody in which the Fab portion that further binds to CD47 comprises LC (Sequence No.: 337) and HC 1367 (Sequence No.: 338).

[0030] In addition, the present invention relates to a pharmaceutical composition for controlling tumor cells, comprising the bispecific entity described herein, to be administered to a patient in need thereof.

[0031] In addition, the present invention relates to a pharmaceutical composition for the treatment of B-cell disorders or B-cell malignancies, comprising the bispecific individual described herein, to be administered to a patient in need thereof.

[0032] The present invention relates to a method for controlling tumor cells, comprising the step of further administering an effective amount of the bispecific entity described herein to a patient in need thereof.

[0033] Additionally, the present invention relates to a method for treating B-cell disorders or B-cell malignancies, comprising the step of administering an effective amount of the bispecific entity described herein to a patient in need thereof. Brief explanation of the drawing

[0034] FIG. 1 is a schematic diagram of specific properties of the bispecific organism described herein engineered to overcome the challenges of universal CD47 expression, including low affinity without binding affinity to CD47; minimal binding to normal cells, i.e., no tissue sink; and high affinity selective binding affinity to CD20 resulting in selective binding to tumor cells. TAA: Tumor-associated antigen Figure 2 illustrates an exemplary bispecific organism structure, protein manipulation features, and various biopharmacological properties. Figures 3a-3c show that the exemplary species bispecific individuals described herein induce macrophage-mediated phagocytosis in CD20+ CD47+ OCI-Ly3 NHL cells. Figures 3a-3b are graphs showing the percentage of phagocytic macrophages according to antibody concentration. Figure 3c is a table showing KD and EC50 values ​​for the bispecific species described herein. Figures 4a-4c show that the CD47xCD20 IgG1 strain described herein, an exemplary bispecific individual, demonstrates CDC function. Figures 4a-4b are graphs showing CDC according to antibody concentration. Figure 4c is a table showing the average EC50 values ​​for TPP-1360, TPP-1362, and rituximab. Figures 5a-5c show that the CD47xCD20 IgG1 strain described herein, an exemplary bispecific organism, demonstrates potent ADCC function in CD20-high NHL cells, i.e., significantly higher than that of rituximab. Figures 5a-5b are graphs showing cytotoxicity according to antibody concentration. Figure 5c is a table showing the CD20 / CD47 ratio. FIG. 6 illustrates the exemplary structure of the bispecific entity described herein as well as the characteristics of a specific example. FIG. 7 shows TPP-1360, an exemplary species bispecific individual described herein that, compared to the binding of TPP-23 (408_437 Fab (VL: SEQ ID: 899; VH: SEQ ID: 900)) with minimal or no binding to platelets or red blood cells, substantially shifts the binding signal to B-cells and somewhat weakly shifts it to T cells, monocytes, and NK cells, thereby exemplifying selective binding to B-cells in human whole blood. Figure 8 is CD47 + / CD20 + It selectively binds to Raji cells but CD47 + / CD20 - TPP-1360 is an exemplary species bispecific individual described herein that has been proven not to bind to human red blood cells (RBCs). FIG. 9 shows that in the co-culture of Raji cells and human RBCs, TPP-1360, an exemplary species bispecific individual described herein, CD47 + / CD20 + It showed dose-dependent binding to Raji cells, but did not bind to human RBCs even at high concentrations of 1 mg / mL. Figure 10 shows, for example, TPP-1360 CD20 + / CD47 + This exemplifies that the binding of recombinant human SIRPα-Fc to human CD47 expressed on the surface of lymphoma cell line OCI-Ly3 was strongly and completely blocked. Figure 11 shows, for example, TPP-1360 CD20 + / CD47 + This exemplifies that the binding of recombinant human SIRPα-Fc to human CD47 expressed on the surface of the lymphoma cell line Raji was strongly and completely blocked. Figure 12 shows, for example, TPP-1360 processing CD20 + This exemplifies the induction of macrophage-mediated phagocytosis in the malignant B cell line Raji. Figure 13 shows, for example, TPP-1360 processing CD20 + This exemplifies the induction of macrophage-mediated phagocytosis in the malignant B cell line OCI-Ly3. Figure 14 shows, for example, TPP-1360 processing CD20 + This exemplifies the induction of macrophage-mediated phagocytosis in the malignant B cell line REC-1. Fig. 15 shows, for example, TPP-1360 processing CD20 + This exemplifies that macrophage-mediated phagocytosis was induced in the malignant B cell line RIVA. Figure 16 shows, for example, that TPP-1360 treatment triggered significantly more efficient phagocytosis in Raji and OCI-Ly3 cells than rituximab, possibly due to the simultaneous blockade of SIRPα-CD47 interactions and the involvement of activating receptors such as FcγR by TPP-1360. Figure 17 shows the binding of rituxan and a bispecific antibody, such as TPP-1360, to Raji cells (CD20+ / CD47+) as measured by surface plasmon resonance (SPR). Figure 18 shows the immunogenicity scale of EpiMatrix antibodies. Figure 19 shows the treatment of TPP-1362 and rituximab in a Raji xenograft model. Figure 20 shows the treatment of TPP-1360 and rituximab in a Raji xenograft model. FIG. 21 shows, for example, TPP-1360 and TPP-1362 CD20 + / CD47 +This exemplifies that recombinant human SIRPα binding to human CD47 expressed on the surface of lymphoma cell line OCI-Ly3 was potently and completely blocked. Rituxan was found not to affect SIRPα binding. Specific details for implementing the invention

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All publications and patents mentioned herein are incorporated by reference.

[0036] As used herein, an article may refer to one or more than one (e.g., at least one) of the grammatical objects of the article.

[0037] As used herein, “approximately” may generally refer to an acceptable degree of error for a given measured quantity, given the attribute or precision of the measurement. An example of a degree of error is within 5% of a given value or range of values.

[0038] An embodiment described herein as “comprising” one or more features may also be construed as the disclosure of a corresponding embodiment that is “consisting” of and / or “essentially composed” of such features.

[0039] "Low affinity for CD47" as used herein refers to an affinity for CD47 of less than about 25 μM, e.g., from about 0.05 μM to about 25 μM, when measured in vitro by SPR as Kd.

[0040] "High affinity for CD20" as used herein refers to an affinity for CD20 of about 0.4 nM or more, e.g., from about 0.4 nM to about 12 nM. In some embodiments, "high affinity for CD20" refers to an affinity for CD20 of about 0.4 nM to about 5 nM.

[0041] The bispecific organism described herein selectively binds to CD47 on tumor cells and substantially has no binding to CD47 on normal cells. As used herein, "substantially has no binding to CD47" generally refers to less than 5% binding to CD47 on (CD20- / CD47+) normal cells. The bispecific organism described herein binds to less than 2% of CD47 on (CD20- / CD47+) normal cells. See Example 5. In other words, the organism described herein generally exhibits more than 95% binding to (CD20+ / CD47+) cells. The organism described herein exhibits more than 98% binding to (CD20+ / CD47+) cells.

[0042] As used herein, the term “pharmaceuticalally acceptable” refers to that which is approved by federal or state regulatory agencies, or is listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally accepted pharmacopoeias for use in animals, and more particularly in humans.

[0043] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in range format. It should also be understood that such range formats are used solely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly mentioned as limits of the range, but also all individual numerical values ​​or sub-ranges included within the range as explicitly mentioned for each numerical value and sub-range.

[0044] Minor variations in the amino acid sequence of the antibody of the present invention are considered to be included by the present invention if the variations in the amino acid sequence(s) maintain at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or 99% sequence homology or identity with respect to the sequence of the antibody or antigen-binding fragment provided herein.

[0045] The antibody of the present invention may comprise a variant in which an amino acid residue from one species is substituted with a corresponding residue from another species at a conserved or non-conserved position. In one embodiment, an amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. In particular, a conserved amino acid substitution is considered.

[0046] As used herein, “conservative amino acid substitution” refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine), have been defined in the art. Therefore, when an amino acid of a polypeptide is replaced by another amino acid from the same side chain family, the amino acid substitution is considered conservative. The inclusion of a conservatively modified variant in the antibody of the present invention does not exclude other forms of variants, e.g., polymorphic variants, interspecies homologs, and alleles.

[0047] As used herein, “non-conservative amino acid substitution” comprises (i) a residue having a positive side chain (e.g., arginine, histidine, or lysine) being substituted with or by a negative residue (e.g., glutamate or aspartate), (ii) a hydrophilic residue (e.g., serine, or threonine) being substituted with or by a hydrophobic residue (e.g., alanine, leucine, isoleucine, phenylalanine, or valine), (iii) a cysteine ​​or proline being substituted with or by any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., valine, histidine, isoleucine, or tryptophan) being substituted with or by a residue having a smaller side chain (e.g., alanine or serine) or a residue without a side chain (e.g., glycine).

[0048] As used herein, the terms “antibody” and “antibodies” refer to polyvalent antibodies including but not limited to conventional isoforms and monospecific forms, as well as currently known bispecific entities in the art, and bispecific antibodies including but not limited to forms otherwise described herein.

[0049] A typical antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light and heavy chains of such antibodies are polypeptides composed of multiple domains. Each heavy chain comprises a heavy chain variable domain (abbreviated as "VH" herein) and a heavy chain constant domain (abbreviated as "CH" herein). The heavy chain constant domain comprises heavy chain constant domains CH1, CH2, and CH3 (antibody classes IgA, IgD, and IgG) and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (abbreviated as "VL" herein) and a light chain constant domain (abbreviated as "CL" herein). The variable domains VH and VL may be further subdivided into hypervariable domains called complementarity determining domains (CDR) interspersed with more conserved domains called framework domains (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminal to carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "constant domains" of the heavy and light chains do not directly involve the binding of the antibody to the target, but exhibit various effector functions.

[0050] Binding between an antibody and its target antigen or epitope is mediated by a complementarity determining region (CDR). A CDR is a highly sequence-variable region located within the variable regions of the antibody heavy and light chains that form the antigen-binding site. The CDR is a major determinant of antigen specificity. Typically, each of the antibody heavy and light chains contains three non-consecutive CDRs. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity / affinity of the antibody according to the present invention and thus provide a further aspect of the present invention.

[0051] Accordingly, the term “antigen-binding fragment” as used herein comprises any naturally occurring or artificially constructed configuration of an antigen-binding polypeptide comprising 1, 2 or 3 light chain CDRs and / or 1, 2 or 3 heavy chain CDRs, wherein the polypeptide can bind to an antigen.

[0052] The sequence of the CDR is any number system known in the art, for example, the Kabat system (Kabat, EA, et al ., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); Chothia system (Chothia &, Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196, 901-917 (1987)); or IMGT system (Lefranc et al ..., can be identified by referring to "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains," Dev. Comp. Immunol. 27, 55-77 (2003)). The CDRs presented herein utilize boundaries, i.e., sizes, according to KABAT. The position numbering of the antibody constant regions described and mentioned herein generally follows KABAT. However, the numbering of the anti-CD47 VL and VH regions described herein, i.e., antibody residue positions and substituted positions, begins with the N-terminal residue of VL or VH, respectively, with reference to each variable region, specifically SEQ ID NO:325 and SEQ ID NO:326, respectively.

[0053] "The bispecific entities described herein" generally refer to the functionally defined antibodies, bispecific element forms, element sequences, antibodies, and antibody species described herein.

[0054] As used herein, the terms “Fab portion” or “arm” refer to the antigen-binding fragment of an antibody, i.e., the region of the antibody that binds to an antigen. As used herein, this comprises one variable domain (VL / VH) of each of the light chain and the heavy chain.

[0055] The "Fab' fragment" contains a single light chain and a single heavy chain, but in addition to CH1 and VH, the "Fab' fragment" contains a heavy chain region between the CH1 and CH2 domains, which requires the formation of an interchain disulfide bond. Thus, two "Fab' fragments" can associate through the formation of a disulfide bond to form an F(ab')2 molecule.

[0056] The "F(ab')2 fragment" contains two light chains and two heavy chains. Each chain contains a portion of an invariant region necessary to form an interchain disulfide bond between the two heavy chains.

[0057] The "Fv fragment" contains only variable regions of the heavy and light chains. It does not contain invariant regions.

[0058] A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0059] A single-stranded Fv ("scFv") is an antibody fragment containing the VH and VL domains of an antibody that are linked together to form a single strand. Polypeptide linkers are typically used to link the VH and VL domains of scFv.

[0060] TandAb ® "Tandem scFv," also known as [another type of scFv], is a single-chain Fv molecule formed by the covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.

[0061] Bispecific T cell engaging antibody (BiTE ® ) is a fusion protein consisting of two single-chain variable fragments (scFv) on a single peptide. One of the scFvs binds to T cells via the CD3 receptor, and the other binds to tumor cell antigens.

[0062] A "diabody" is a small divalent and bispecific antibody fragment comprising a heavy chain (VH) variable domain (VH-VL) connected to a light chain variable domain (VL) on the same polypeptide chain by a very short peptide linker to allow pairing between two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with a complementary domain on another chain and promotes the assembly of a dimeric molecule having two functional antigen-binding sites.

[0063] "DARPin" is a bispecific ankyrin repeat molecule. DARPin is derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. DARPin library modules are defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for initial design and another 2,200 for subsequent refinement. The modules serve as building blocks for the DARPin library. The library modules are similar to the human genome sequence. DARPin consists of 4 to 6 molecules. Since each module is approximately 3.5 kDa, the average DARPin size is 16–21 kDa. The selection of binders is performed by completely cell-free ribosomal display and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0064] As used herein, the terms “tumor” and “tumor cell” broadly refer to cancer cells, including but not limited to cells undergoing abnormal proliferation, hematological oncological conditions, hematological malignancies, lymphoproliferative disorders, B-cell disorders, B-cell malignancies, and B-cell lymphomas.

[0065] As used herein, the IgG1 or IgG1 1+1 heterodimer form basically refers to a total IgG1 antibody composed of (i) one source, namely one heavy chain (HC) and one light chain (LC) on one side of anti-CD47; and another source, for example, one heavy chain (HC) and one light chain (LC) on the other side of anti-CD20. See, for example, FIGS. 2 and FIGS. 6.

[0066] CD47

[0067] The value of cancer immunotherapy targeting the CD47 / SIRPα axis is widely established. For example, Weiskopf, K., et al ., Eur J Cancer. 2017 May;76:100; Feng, M.; et al See Nat Rev Cancer. 2019 Oct;19(10):568-586. Anti-CD47 approaches in the clinic have been limited by the need for combination therapy, tissue sinks targeting CD47 with high-affinity binders, immunogenicity, and hematological toxicity (anemia, neutropenia, and / or thrombocytopenia) observed with some clinical molecules.

[0068] CD47 is upregulated on tumor cells, but it is also universally expressed at relatively high levels on all cells, including NK cells, RBCs, and platelets. Therefore, monospecific agonists targeting CD47 tend to exhibit poor pharmacokinetic properties due to side effects including target-mediated drug batching (TMDD), anemia, and thrombocytopenia. Target-mediated drug batching (TMDD) is a phenomenon in which a drug binds with high affinity to a pharmacological target site (e.g., a receptor) to the extent that it affects pharmacokinetic properties. Anti-CD47 IgG4 mAbs are generally required to reduce toxicity. Consequently, the activity of a single anti-CD47, e.g., IgG1 agonist tends to be limited.

[0069] Russ, A., et al ., Blood Rev. 2018 Nov;32(6):480 describes a specific and widely characterized IgG4 anti-CD47 antibody (CC-90002). In particular, see WO2013119714 (US Patent No. 9045541). Various amino acid substitutions in this antibody, designed to increase cell-free production and decrease immunogenicity, have been described. See WO2016109415 (US20170369572); WO2018009499 (US20190241654); and WO2018183182. CC-90002 is a high-affinity IgG4 P / E anti-CD47 molecule that binds to CD47 expressed in diseased and normal tissues.

[0070] However, because CD47 is widely expressed in tumor cells as well as normal cells, high-affinity anti-CD47 antibodies may cause undesirable toxicity. Accordingly, the present invention provides a bispecific antibody comprising a CD47-binding domain that is improved over CC-90002 in terms of toxicity and efficacy. In particular, the bispecific entity described herein selectively and safely targets tumor cells with little to no binding to CD47 in peripheral tissues. The present invention relates to an antibody comprising at least one Fab portion binding to CD47 and at least one Fab portion binding to CD20; wherein the Fab portion binding to CD47 exhibits low affinity for CD47 (e.g., Kd greater than 100 nM); wherein the Fab portion binding to CD20 exhibits high affinity for CD20 (e.g., Kd less than 5 nM); wherein the bispecific antibody selectively binds to CD47 in tumor cells and has substantially no binding to CD47 in normal cells.

[0071] CC-90002 is provided as a source of anti-CD47 elements for the construction of a reference parent sequence and some of the bispecific entities described herein. CC-90002 VL CDRs are SEQ ID NO: 347 (CDRL1), SEQ ID NO: 348 (CDRL2), and SEQ ID NO: 349 (CDRL3). CC-90002 VH CDRs are SEQ ID NO: 350 (CDRH1), SEQ ID NO: 351 (CDRH2), and SEQ ID NO: 352 (CDRH3). CC-90002 VL (SEQ ID NO: 325) and VH (SEQ ID NO: 326) are also provided for reference. CC-90002 VL fused to a natural IgG1 LC constant region to form a whole LC for reference is provided as CC-90002 whole LC / IgG1 (SEQ ID NO: 327). CC-90002 VH fused to a natural IgG1 HC constant region to form a whole HC for reference is provided as CC-90002 whole HC / IgG1 (SEQ ID NO: 328).

[0072] Both the VH and VL regions of CC-90002 were engineered to reduce immunogenicity, while maintaining functionality for use in the bispecific individuals described herein.

[0073] For example, the bispecific entities described and exemplified herein are demonstrated to overcome the challenges of monoagonist anti-CD47 mAb therapy. Due to their affinity for detuned CD47, the CD47xCD20 bispecific entities described and provided herein [are] CD20 + It preferentially binds to tumor-associated antigen (TAA) cells, CD47 + It reduces cell-mediated sink effects and extra-target toxicity. In addition, cellular efficacy, in vivo efficacy, and safety data indicate that the CD47xCD20 bispecific agent described and exemplified herein provides a unique option as a monotherapy for, for example, CD20-positive B-cell malignancies.

[0074] FIG. 1 is a schematic diagram of specific properties of the bispecific organism described herein engineered to overcome the challenges of universal CD47 expression, including low affinity without binding affinity to CD47; minimal binding to normal cells, i.e., no tissue sink; high affinity selective binding affinity to the tumor-associated antigen (TAA) CD20; which results in selective binding to tumor cells.

[0075] The CD47xCD20 bispecific entities provided herein may comprise a CD20 binding domain from any high-affinity CD20 binder. Rituximab LC (SEQ: 329) and HC (SEQ: 330) are preferred sources of anti-CD20 elements for constructing the bispecific entities described herein. In certain embodiments, the bispecific antibodies provided herein comprise one or both of rituximab VL (SEQ: 323) and VH (SEQ: 324), or each comprises rituximab VL CDR: SEQ: 353 (CDRL1), SEQ: 354 (CDRL2), and SEQ: 355 (CDRL3); and rituximab VH CDR: SEQ: 356 (CDRH1), SEQ: 357 (CDRH2), and SEQ: 358 (CDRH3). The anti-CD20 IgG1 LC and HC constant regions as otherwise described herein are fused to the carboxyl terminus of rituximab VL (SEQ No.: 323) and rituximab VH (SEQ No.: 324), respectively. Anti-CD20 LC (SEQ No.: 331) is preferred for use in constructing the bispecific organism of the present invention. Anti-CD20 HC (SEQ No.: 332) is preferred for use in constructing the bispecific organism of the present invention.

[0076] The exemplary bispecific organism described herein has substantially no binding to CD47 in peripheral tissues, RBCs, and platelets and selectively and safely targets CD+ tumor cells, providing an extended half-life of low toxicity, wherein the antibody mediates complement-dependent cytotoxicity (CDC) of tumor cells.

[0077] The bispecific individual is preferably in the form of an IgG1 1+1 heterodimer.

[0078] In certain embodiments, the bispecific antibody provided herein comprises two antigen-binding arms covalently linked to form a single entity. IgG bispecific antibodies utilizing two Fab domains require careful consideration regarding expression and purification strategies to ensure proper assembly of the desired product. Efforts may be made to bias the expression of the desired bispecific antibody at the genetic level. For example, substitution in the CH3 domain of IgG Fc has been described to induce heteromerization of Fc. Additionally, a knob-in-hole strategy utilizes steric hindrance to create a complementary asymmetric molecular plane between two different Fc CH3 domains. Other strategies known in the art utilize electrostatic complementarity to induce specificity. Alternatively, a wild-type IgG scaffold may be used, and the resulting product assembly may be separated during protein purification to isolate the desired product.

[0079] A preferred bispecific antibody of the present invention is a natural human IgG1 antibody composed basically of (A) one anti-CD47 IgG1 (monomeric) portion containing one whole light chain (LC) and one whole heavy chain (HC), as well as (B) one anti-CD20 IgG1 (monomeric) portion containing one whole light chain (LC) and one whole heavy chain (HC). The two monomers form a conventional dimeric IgG1 antibody, wherein one arm (Fab1) of CD47 weakened While providing binding, the other arm (Fab2) provides affinity binding and binding strength to CD20. FIGS. 2 and FIGS. 6 illustrate the exemplary structure and exemplary protein manipulation features of CD47 x CD20 described herein.

[0080] The preferred monomeric elements of the IgG1 1+1 heterodimer described herein each contain specific subsequences within LC and HC invariant regions that reduce homodimer formation during the production of the IgG1 1+1 heterodimer format. In one embodiment, the anti-CD47 LC invariant region that ensures proper LC / HC pairing during production comprises SEQ ID NO: 340. In another embodiment, the anti-CD47 HC invariant region that ensures Fc heterodimer formation during production comprises SEQ ID NO: 342. The preferred anti-CD20 LC invariant region that ensures proper LC / HC pairing during production comprises SEQ ID NO: 344. The preferred anti-CD20 HC invariant region that ensures Fc heterodimer formation during production comprises SEQ ID NO: 346. An exemplary anti-CD47 LC invariant region that ensures proper LC / HC pairing during production is SEQ ID NO: 339. An exemplary anti-CD47 HC constant region that ensures Fc heteromer formation during production is SEQ ID NO: 341. An exemplary anti-CD20 LC constant region that ensures proper LC / HC pair formation during production is SEQ ID NO: 343. An exemplary anti-CD20 HC constant region that ensures Fc heteromer formation during production is SEQ ID NO: 345.

[0081] Fab of CD47 weakened The preferred IgG1 constant regions for use with the 161 Fabs described herein in the IgG1 1+1 heteromer format providing binding are the LC constant region SEQ NO:339 and the HC constant region SEQ NO:341.

[0082] Compared to CC-90002, an anti-CD47 IgG1 LC constant region is provided that includes substitutions Q124E, L135W, Q160E, and T180E, which ensure proper LC / HC pair formation during the production of the IgG1 1+1 heterodimer format.

[0083] The present invention further provides a preferred anti-CD47 IgG1 LC constant region (SEQ ID NO: 339). In a specific embodiment, e.g., a specific embodiment of the bispecific antibody provided herein, the anti-CD47 VL region provided herein is fused to the amino terminus of SEQ ID NO: 339. SEQ ID NO: 340 is located within SEQ ID NO: 339. SEQ ID NO: 339 and SEQ ID NO: 440 comprise substitutions Q124E, L135W, Q160E, and T180E, which ensure proper LC / HC pairing during the production of the IgG1 1+1 heterodimer format.

[0084] An anti-CD47 IgG1 HC constant region is provided, comprising substitutions Q179K, T371V, T389L, K420L, and T422W, which ensure proper LC / HC pair formation and Fc heterodimer formation during the production of IgG1 1+1 heterodimer form.

[0085] The present invention further provides a suitable anti-CD47 IgG1 HC constant region (SEQ ID NO: 341). In a specific embodiment, e.g., a specific embodiment of the bispecific antibody provided herein, the anti-CD47 VH region provided herein is fused to the amino terminus of SEQ ID NO: 341. SEQ ID NO: 342 is located within SEQ ID NO: 341. SEQ ID NO: 341 and SEQ ID NO: 342 comprise substitutions Q179K, T371V, T389L, K420L, and T422W that ensure the formation of an Fc heterodimer during the production of an IgG1 1+1 heterodimer format.

[0086] Compared to rituximab, an anti-CD20 IgG1 LC constant region is provided that includes substitutions F116A, Q124R, L135V, and T178R, which ensure proper LC / HC pair formation during the production of the IgG1 1+1 heterodimer format.

[0087] The present invention further provides a preferred anti-CD20 IgG1 LC constant region (SEQ ID NO: 343). In a specific embodiment, e.g., a specific embodiment of the bispecific antibody provided herein, the anti-CD20 VL region provided herein is fused to the amino terminus of SEQ ID NO: 343. SEQ ID NO: 344 is located within SEQ ID NO: 343. SEQ ID NO: 343 and SEQ ID NO: 344 comprise substitutions F116A, Q124R, L135V, and T178R that reduce homodimer formation during the production of the IgG1 1+1 heterodimer format.

[0088] Compared to rituximab, an anti-CD20 IgG1 HC constant region is provided that includes substitutions A139W, L143E, K145T, Q179E, T371V, L372Y, F436A, and Y438V that ensure the formation of Fc heterodimers during the production of IgG1 1+1 heterodimer forms.

[0089] The present invention further provides a suitable anti-CD20 IgG1 HC constant region (SEQ ID NO: 345). In a specific embodiment, e.g., a specific embodiment of the bispecific antibody provided herein, the anti-CD20 VH region provided herein is fused to the amino terminus of SEQ ID NO: 345. SEQ ID NO: 346 is located within SEQ ID NO: 345. SEQ ID NO: 345 and SEQ ID NO: 346 comprise substitutions A139W, L143E, K145T, Q179E, T371V, L372Y, F436A, and Y438V that reduce homodimer formation during the production of the IgG1 1+1 heterodimer form.

[0090] CD47xCD20 bispecific entities

[0091] The CD47xCD20 bispecific program was initiated to identify therapeutic antibodies capable of blocking human CD47 binding to SIRPα only on CD20-expressing cells. Examples generated from that project and provided herein bind to CD20 with high affinity while exhibiting uncoordinated affinity for CD47. Once bound to CD20 on tumor cells, the antibody potently blocks CD47-SIRPα interactions and co-engages with the activating receptor FcγR on effector cells via IgG1 Fc, resulting in macrophage-mediated phagocytosis and the activation of natural killer (NK) cell-mediated cytotoxicity against tumor cells.

[0092] Bispecific antibody containing anti-CD47 derived from CC-90002

[0093] CD47 Epitope Mapping and CC-90002

[0094] The anti-CD47 epitope was determined by determining the crystal structure of the uncoordinated parent version of CC-90002 (408_437) Fab (VL: SEQ ID: 387; VH: SEQ ID: 388) as a complex with the human CD47 extracellular domain at a resolution of 2.4 Å. All three light chain (LC) CDRs (SEQ ID: 347, SEQ ID: 348, SEQ ID: 349) and heavy chain (HC) CDR2 (SEQ ID: 351) and HC CDR3 (SEQ ID: 352) participate in binding to the large surface area of ​​the CD47 ECD. The HC CDRs make multiple contacts with the KGRD loop of CD47, and the LC CDRs overlap with the SIRPα binding site, which explains the ability of CC-90002 and the bispecific entity described herein to block SIRPα binding.

[0095] The CD47XCD20 bispecific individual described herein is CD47 + / CD20 -It is designed to promote CD20-restricted blockade of the CD47-SIRPα "don't eat me" signal on cancer cells expressing both CD20 and CD47 while preserving normal cells. Multiple steps of protein manipulation resulted in the anti-CD47 Fab of the bispecific organism described herein. (1) Protein manipulation utilized both the VH and VL chains of CC-90002 to reduce immunogenicity while maintaining functionality; and (2) Protein manipulation also utilized unmodified CC-90002 to demodify it. The IgG1 bispecific antibody targeting CD47 and CD20 with reduced affinity for CD47 described and exemplified herein 1) maintains efficacy mediating anti-tumor function by targeting CD47-SIRPα interactions and engaging the activating receptor FcγR; and 2) minimizes target-mediated sink effects and toxicity observed with anti-CD47 therapeutics; 3) Avoid the need for combination therapy using two monoclonal antibodies, including CD47 and CD20 interventions in a single molecule.

[0096] Protein Design

[0097] The crystal structure of the effector antigen CD47 was bound to the high-affinity anti-CD47 Fab (CC-90002 (408_437 VL: SEQ No.: 899; 408_437 VH: SEQ No.: 900)), leading to the construction of an in silico library of Fab variants predicted to have a low affinity range, excellent stability, and low immunogenicity. Variants from this library were expressed as IgG1 bispecific individuals with the high-affinity anti-CD20 Fab (rituximab (VL: SEQ No.: 323; VH: SEQ No.: 324)).

[0098] 143 generated physical constructs were screened for selectivity and efficacy using cell-based assays for CD47 binding and SIRPα blockade to identify variants that effectively bind to the effector antigen CD47 on target cells co-expressing the selector antigen CD20, but bind minimally to non-target cell lines expressing only the effector antigen CD47.

[0099] Exemplary anti-CD47 whole LC and HC, LC and HC constant regions, VL and VH regions, and CDR sequences having substantially reduced binding affinity and reduced immunogenicity for CD47 compared to CC-90002, including but not limited to IgG1 isoforms, are provided and otherwise described herein. As described below, an IgG1 1+1 heterodimer form is preferred, comprising (A) one anti-CD47 IgG1 (monomeric) portion containing one whole light chain (LC) and one heavy chain (HC), as well as (B) one anti-CD20 IgG1 (monomeric) portion containing one whole light chain (LC) and one whole heavy chain (HC).

[0100] The antibody of the present invention comprises VL and VH amino acid sequences derived from CC-90002, namely SEQ ID NO: 325 and SEQ ID NO: 326, respectively, wherein the binding affinity for CD47 is substantially attenuated; that is, the Fab portion binding to CD47 exhibits low affinity. Screening of the uncoordinated anti-CD47 binders described herein revealed that while the affinity range for CD47 showed a dramatic decrease in binding to non-target cells, they were still able to effectively bind to CD47 in a recessive manner when recruited to the surface of target cells after binding to CD20. The initial uncoordinated CD47 x CD20 bispecific read achieved this selectivity with an affinity range of about 0.5 μM to about 2.5 μM. FIG. 3a-3c. For example, TPP-1360 was measured to have an affinity of 1.7 μM Kd for human CD47 ECD, which reflects a reduction of about 350-fold in affinity compared to the parent anti-CD47 binder. TPP-1362 was measured to have an affinity of 0.796 μM Kd for human CD47 ECD, which reflects a reduction of about 150-fold in affinity compared to the parent anti-CD47 binder. The uncoordinated CD47 x CD20 bispecific individuals described herein exhibit selectivity with affinities ranging from about 0.2 μM to about 4 μM.

[0101] The bispecific organism described herein selectively binds to CD47 on CD20 expressing tumor cells and substantially has no binding to CD47 in normal cells. In a co-culture binding assay for the bispecific organism described herein, the binding ratio for Raji (CD47+CD20+) versus human RBCs is, for example, about 6,000-fold. The binding ratio for the bispecific organism described herein for human B cells (CD47+CD20+) versus human RBCs is, for example, about 700-fold. The selection level of the bispecific organism described herein exhibits a selection range of about 400 to about 8,000-fold, depending on the expression levels of CD20 and CD47 on tumor cells and normal cells. Therefore, assuming a fixed level of CD47 expression, the bispecific organism described herein exhibits increased selectivity and efficacy as the CD20 level increases.

[0102] An exemplary antibody of the present invention also comprises VL and VH regions derived from CC-90002, namely SEQ ID NO: 325 and SEQ ID NO: 326, respectively, wherein VL, VH, or both comprise one or more amino acid substitutions that substantially reduce the immunogenicity of the generated antibody. An exemplary antibody of the present invention comprises VL and VH regions derived from CC-90002, namely SEQ ID NO: 325 and SEQ ID NO: 326, respectively, wherein the sequences of VL, VH, or both comprise one or more amino acid substitutions that reduce the binding affinity of the generated antibody to CD47 and substantially reduce the immunogenicity of the generated antibody.

[0103] Accordingly, the preferred antibody derived from CC-90002 described herein exhibits 1-7 amino acid substitutions for VL SEQ ID NO:325; and 1-11 amino acid substitutions for VH SEQ ID NO:326.

[0104] An antibody derived from CC-90002 having 8 amino acid substitutions for VL SEQ ID NO:325 is further considered and otherwise functionally described herein. An antibody derived from CC-90002 having 9 amino acid substitutions for VL SEQ ID NO:325 is further considered and otherwise functionally described herein. An antibody derived from CC-90002 having 10 amino acid substitutions for VL SEQ ID NO:325 is further considered and otherwise functionally described herein.

[0105] An antibody derived from CC-90002 having 12 amino acid substitutions for VH SEQ ID NO:326 is further considered and otherwise functionally described herein. An antibody derived from CC-90002 having 13 amino acid substitutions for VH SEQ ID NO:326 is further considered and otherwise functionally described herein. An antibody derived from CC-90002 having 14 amino acid substitutions for VH SEQ ID NO:326 is further considered and otherwise functionally described herein.

[0106] The present invention provides a CD47 antibody in which anti-CD47 VL represents 1 to 7 amino acid substitutions for SEQ ID NO:325, wherein at least one of the amino acid substitutions is selected from the group consisting of A10S, M11L, K24R, A51E, N52S, L54F, and S56D; and anti-CD47 VH represents 1 to 11 amino acid substitutions for SEQ ID NO:326, wherein at least one of the amino acid substitutions is selected from the group consisting of T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L.

[0107] The present invention also provides a CD47 antibody in which the anti-CD47 VL exhibits at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acid substitutions for SEQ ID NO:325, wherein the amino acid substitutions are selected from the group consisting of A10S, M11L, K24R, A51E, N52S, L54F, and S56D.

[0108] The present invention relates to the antibody described herein, wherein anti-CD47 VL exhibits at least 7 amino acid substitutions for SEQ ID NO:325, wherein the 7 amino acid substitutions are A10S, M11L, K24R, A51E, N52S, L54F, and S56D.

[0109] The present invention relates to the antibody described herein, wherein the anti-CD47 VH represents at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions for SEQ ID NO:326, wherein the amino acid substitutions are selected from the group consisting of T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L.

[0110] The present invention relates to the antibody described herein, wherein anti-CD47 VH exhibits at least 11 amino acid substitutions for SEQ ID NO:326, wherein the 11 amino acid substitutions are T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L.

[0111] Anti-CD47 LC (SEQ No. 335) and HC (SEQ No. 336) are preferred sources of anti-CD47 elements for the construction of VL (SEQ No. 319) and VH (SEQ No. 320), comprising the bispecific entities described herein, in particular VL CDR SEQ No. 365 (CDRL1), SEQ No. 366 (CDRL2), and SEQ No. 367 (CDRL3), respectively; and VH CDR SEQ No. 368 (CDRH1), SEQ No. 369 (CDRH2), and SEQ No. 370 (CDRH3). Substitutions A10S, M11L, K24R, A51E, N52S, L54F, and S56D are important VL (SEQ No. 319) substitutions associated with CC-90002 that affect low CD47 binding affinity and reduce immunogenicity. T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L are VHs (SEQN: 320) that are important in relation to CC-90002, which affect low CD47 binding affinity (particularly E59Y and S102E) and reduce immunogenicity. Anti-CD47 IgG1 LC and HC invariant regions as otherwise described herein, e.g., SEQN: 339 and SEQN: 341, respectively, are fused to the carboxyl ends of VL (SEQN: 319) and VH (SEQN: 320) in certain embodiments. Anti-CD47 LC (SEQN: 335) is preferred for use in the construction of the bispecific organism of the present invention. Anti-CD47 HC (SEQ: 336) is preferred for use in the construction of the bispecific organism of the present invention. Substitutions Q124E, L135W, Q160E, and T180E are important SEQ: 335 positions that ensure proper LC / HC pair formation during the production of the IgG1 1+1 heterodimer format. Substitutions Q179K, T371V, T389L, K420L, and T422W are important SEQ: 336 positions that reduce the tendency toward homodimer formation during the production of the IgG1 1+1 heterodimer format.

[0112] The present invention further comprises, in particular, anti-CD47 VL having 1 to 3 amino acid substitutions for SEQ ID NO: 325, wherein at least 1, at least 2, or at least 3 amino acid substitutions in anti-CD47 VL (SEQ ID NO: 325) are selected from the group consisting of A10S, M11L, and K24R (e.g., SEQ ID NO: 321); The present invention relates to an antibody otherwise described herein, wherein anti-CD47 VH exhibits 1 to 11 amino acid substitutions for SEQ ID NO: 326, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acid substitutions in anti-CD47 VH (SEQ ID NO: 326) are selected from the group consisting of T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L (e.g., SEQ ID NO: 322).

[0113] The present invention relates particularly to an antibody otherwise described herein, wherein anti-CD47 VH exhibits 1 to 11 amino acid substitutions for SEQ ID NO: 326, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acid substitutions in anti-CD47 VH (SEQ ID NO: 326) are selected from the group consisting of T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L (e.g., SEQ ID NO: 322).

[0114] Anti-CD47 LC (SEQN: 337) and HC (SEQN: 338) are preferred sources of anti-CD47 elements for the construction of the bispecific entities described herein, particularly VL (SEQN: 321) and VH (SEQN: 322), each comprising VL CDRs: SEQN: 371, SEQN: 372, and SEQN: 373; and VH CDRs: SEQN: 374, SEQN: 375, and SEQN: 376. A10S, M11L, and K24R are important VL (SEQN: 321) sites that affect low CD47 binding affinity and reduce immunogenicity. Substitutions T14P, Q43K, A44G, E59Y, D66G, M76T, S84R, S88A, M93V, S102E, and T115L are important VH (SEQN: 322) sites that affect low CD47 binding affinity (particularly E59Y and S102E) and reduce immunogenicity. Anti-CD47 IgG1 LC and HC invariant regions as otherwise described herein, e.g., SEQN: 339 and SEQN: 341, respectively, are fused to the carboxyl ends of VL (SEQN: 321) and VH (SEQN: 322). Anti-CD47 LC (SEQN: 337) is preferred for use in the construction of the bispecific organism of the present invention. Anti-CD47 HC (SEQN: 338) is preferred for use in the construction of the bispecific organism of the present invention. Substitutions Q124E, L135W, Q160E, and T180E are important positions at SEQ ID NO:337 for reducing the tendency toward homodimer formation during the production of the IgG1 1+1 heterodimer format. Substitutions Q179K, T371V, T389L, K420L, and T422W are important positions at SEQ ID NO:338 for reducing the tendency toward homodimer formation during the production of the IgG1 1+1 heterodimer format.

[0115] The present invention particularly relates to an anti-CD47 VL having 1 to 10 amino acid substitutions for SEQ ID NO: 325, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions in the anti-CD47 VL (SEQ ID NO: 325) are selected from the group consisting of A10S, M11L, K24Q, K39D, K42T, K45Q, A51E, N52S, L54F, and S56D (e.g., SEQ ID NO: 317); The present invention relates to an antibody otherwise described herein, wherein anti-CD47 VH exhibits 1 to 11 amino acid substitutions for SEQ ID NO: 326, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acid substitutions in anti-CD47 VH (SEQ ID NO: 326) are selected from the group consisting of T14P, A44G, E59Y, D66G, M76T, S84A, R87T, S88A, M93V, S102E, and T115L (e.g., SEQ ID NO: 318).

[0116] The present invention relates particularly to an antibody otherwise described herein, wherein anti-CD47 VH exhibits 1 to 11 amino acid substitutions for SEQ ID NO: 326, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acid substitutions in anti-CD47 VH (SEQ ID NO: 326) are selected from the group consisting of T14P, A44G, E59Y, D66G, M76T, S84A, R87T, S88A, M93V, S102E, and T115L (e.g., SEQ ID NO: 318).

[0117] Anti-CD47 LC (SEQ No. 333) and HC (SEQ No. 334) are preferred sources of anti-CD47 elements for the construction of VL (SEQ No. 317) and VH (SEQ No. 318), comprising the bispecific entities described herein, in particular VL CDRs: SEQ No. 359, SEQ No. 360, and SEQ No. 361; and VH CDRs: SEQ No. 362, SEQ No. 363, and SEQ No. 364, respectively. Substitutions A10S, M11L, K24Q, K39D, K42T, K45Q, A51E, N52S, L54F, and S56D are important VL (SEQ No. 317) positions that affect low CD47 binding affinity and reduce immunogenicity. Substitutions T14P, A44G, E59Y, D66G, M76T, S84A, R87T, S88A, M93V, S102E, and T115L are important VH (SEQN: 318) sites that affect low CD47 binding affinity (particularly E59Y and S102E) and reduce immunogenicity. Anti-CD47 IgG1 LC and HC invariant regions as otherwise described herein, e.g., SEQN: 339 and SEQN: 341, respectively, are fused to the carboxyl ends of VL (SEQN: 317) and VH (SEQN: 318). Anti-CD47 LC (SEQN: 333) is preferred for use in the construction of the bispecific organism of the present invention. Anti-CD47 HC (SEQN: 334) is preferred for use in the construction of the bispecific organism of the present invention. Substitutions Q124E, L135W, Q160E, and T180E are important positions (SEVIN:333) that reduce the tendency for homodimer formation during the generation of the IgG1 1+1 heterodimer format. Substitutions Q179K, T371V, T389L, K420L, and T422W are important positions (SEVIN:334) for reducing the tendency for homodimer formation during the generation of the IgG1 1+1 heterodimer format.

[0118] Exemplary antibodies described and considered herein include anti-CD20 VL CDR RASSSVSYIH (Sequence No.: 353), ATSNLAS (Sequence No.: 354), and QQWTSNPPT (Sequence No.: 355); and, VH CDR SYNMH (Sequence No.: 356), AIYPGNGDTSYNQKFKG (Sequence No.: 357), and STYYGGDWYFNV (Sequence No.: 358). Exemplary bispecific antibodies otherwise described herein include anti-CD20 VL (Sequence No.: 323) and VH (Sequence No.: 324). Exemplary preferred species bispecific antibodies functionally described herein include anti-CD20 LC (Sequence No.: 331) and anti-CD20 HC (Sequence No.: 332).

[0119] 161 examples of the unadjusted term-CD47 VL / VH Fab derived from CC-90002

[0120] The present invention further provides 161 VL and VH Fabs derived from the parent antibody CC-90002. For each of the 161 Fabs, the VL amino acid sequence is provided as odd SEQ ID NOs 1-321; and the VH amino acid sequence is provided as even SEQ ID NOs 2-322. Each identified Fab (VL / VH pair) is numbered with adjacent SEQ ID NOs, i.e., pairs disclosed herein following the following pattern: SEQ ID NO:1 / SEQ ID NO:2; SEQ ID NO:3 / SEQ ID NO:4, etc. to SEQ ID NO:321 / SEQ ID NO:322:

[0121] Sequence No.:1 / Sequence No.:2; Sequence No.:3 / Sequence No.:4; Sequence No.:5 / Sequence No.:6; Sequence No.:7 / Sequence No.:8; Sequence No.:9 / Sequence No.:10; Sequence No.:11 / Sequence No.:12; Sequence No.:13 / Sequence No.:14; Sequence No.:15 / Sequence No.:16; Sequence No.:17 / Sequence No.:18; Sequence No.:19 / Sequence No.:20; Sequence No.:21 / Sequence No.:22; Sequence No.:23 / Sequence No.:24; Sequence No.:25 / Sequence No.:26; Sequence No.:27 / Sequence No.:28; Sequence No.:29 / Sequence No.:30; Sequence No.:31 / Sequence No.:32; Sequence No.:33 / Sequence No.:34; Sequence No.:35 / Sequence No.:36; Sequence No.:37 / Sequence No.:38; Sequence No.:39 / Sequence No.:40; Sequence No.:41 / Sequence No.:42; Sequence No.:43 / Sequence No.:44; Sequence No.:45 / Sequence No.:46; Sequence No.:47 / Sequence No.:48; Sequence No.:49 / Sequence No.:50; Sequence No.:51 / Sequence No.:52; Sequence No.:53 / Sequence No.:54; Sequence No.:55 / Sequence No.:56; Sequence No.:57 / Sequence No.:58; Sequence No.:59 / Sequence No.:60; Sequence No.:61 / Sequence No.:62; Sequence No.:63 / Sequence No.:64; Sequence No.:65 / Sequence No.:66; Sequence No.:67 / Sequence No.:68; Sequence No.:69 / Sequence No.:70; Sequence No.:71 / Sequence No.:72; Sequence No.:73 / Sequence No.:74; Sequence No.:75 / Sequence No.:76; Sequence No.:77 / Sequence No.:78; Sequence No.:79 / Sequence No.:80; Sequence No.:81 / Sequence No.:82; Sequence No.:83 / Sequence No.:84; Sequence No.:85 / Sequence No.:86; Sequence No.:87 / Sequence No.:88; Sequence No.:89 / Sequence No.:90; Sequence No.:91 / Sequence No.:92; Sequence No.:93 / Sequence No.:94; Sequence No.:95 / Sequence No.:96; Sequence No.:97 / Sequence No.:98; Sequence No.:99 / Sequence No.:100; Sequence No.:101 / Sequence No.:102; Sequence No.:103 / Sequence No.:104; Sequence No.:105 / Sequence No.:106; Sequence No.:107 / Sequence No.:108; Sequence No.:109 / Sequence No.:110; Sequence No.:111 / Sequence No.:112; Sequence No.:113 / Sequence No.:114; Sequence No.:115 / Sequence No.:116;Sequence No.:117 / Sequence No.:118; Sequence No.:119 / Sequence No.:120; Sequence No.:121 / Sequence No.:122; Sequence No.:123 / Sequence No.:124; Sequence No.:125 / Sequence No.:126; Sequence No.:127 / Sequence No.:128; Sequence No.:129 / Sequence No.:130; Sequence No.:131 / Sequence No.:132; Sequence No.:133 / Sequence No.:134; Sequence No.:135 / Sequence No.:136; Sequence No.:137 / Sequence No.:138; Sequence No.:139 / Sequence No.:140; Sequence No.:141 / Sequence No.:142; Sequence No.:143 / Sequence No.:144; Sequence No.:145 / Sequence No.:146; Sequence No.:147 / Sequence No.:148; Sequence No.:149 / Sequence No.:150; Sequence No.:151 / Sequence No.:152; Sequence No.:153 / Sequence No.:154; Sequence No.:155 / Sequence No.:156; Sequence No.:157 / Sequence No.:158; Sequence No.:159 / Sequence No.:160; Sequence No.:161 / Sequence No.:162; Sequence No.:163 / Sequence No.:164; Sequence No.:165 / Sequence No.:166; Sequence No.:167 / Sequence No.:168; Sequence No.:169 / Sequence No.:170; Sequence No.:171 / Sequence No.:172; Sequence No.:173 / Sequence No.:174; Sequence No.:175 / Sequence No.:176; Sequence No.:177 / Sequence No.:178; Sequence No.:179 / Sequence No.:180; Sequence No.:181 / Sequence No.:182; Sequence No.:183 / Sequence No.:184; Sequence No.:185 / Sequence No.:186; Sequence No.:187 / Sequence No.:188; Sequence No.:189 / Sequence No.:190; Sequence No.:191 / Sequence No.:192; Sequence No.:193 / Sequence No.:194; Sequence No.:195 / Sequence No.:196; Sequence No.:197 / Sequence No.:198; Sequence No.:199 / Sequence No.:200; Sequence No.:201 / Sequence No.:202; Sequence No.:203 / Sequence No.:204; Sequence No.:205 / Sequence No.:206; Sequence No.:207 / Sequence No.:208; Sequence No.:209 / Sequence No.:210; Sequence No.:211 / Sequence No.:212; Sequence No.:213 / Sequence No.:214; Sequence No.:215 / Sequence No.:216; Sequence No.:217 / Sequence No.:218; Sequence No.:219 / Sequence No.:220;Sequence No.:221 / Sequence No.:222; Sequence No.:223 / Sequence No.:224; Sequence No.:225 / Sequence No.:226; Sequence No.:227 / Sequence No.:228; Sequence No.:229 / Sequence No.:230; Sequence No.:231 / Sequence No.:232; Sequence No.:233 / Sequence No.:234; Sequence No.:235 / Sequence No.:236; Sequence No.:237 / Sequence No.:238; Sequence No.:239 / Sequence No.:240; Sequence No.:241 / Sequence No.:242; Sequence No.:243 / Sequence No.:244; Sequence No.:245 / Sequence No.:246; Sequence No.:247 / Sequence No.:248; Sequence No.:249 / Sequence No.:250; Sequence No.:251 / Sequence No.:252; Sequence No.:253 / Sequence No.:254; Sequence No.:255 / Sequence No.:256; Sequence No.:257 / Sequence No.:258; Sequence No.:259 / Sequence No.:260; Sequence No.:261 / Sequence No.:262; Sequence No.:263 / Sequence No.:264; Sequence No.:265 / Sequence No.:266; Sequence No.:267 / Sequence No.:268; Sequence No.:269 / Sequence No.:270; Sequence No.:271 / Sequence No.:272; Sequence No.:273 / Sequence No.:274; Sequence No.:275 / Sequence No.:276; Sequence No.:277 / Sequence No.:278; Sequence No.:279 / Sequence No.:280; Sequence No.:281 / Sequence No.:282; Sequence No.:283 / Sequence No.:284; Sequence No.:285 / Sequence No.:286; Sequence No.:287 / Sequence No.:288; Sequence No.:289 / Sequence No.:290; Sequence No.:291 / Sequence No.:292; Sequence No.:293 / Sequence No.:294; Sequence No.:295 / Sequence No.:296; Sequence No.:297 / Sequence No.:298; Sequence No.:299 / Sequence No.:300; Sequence No.:301 / Sequence No.:302; Sequence No.:303 / Sequence No.:304; Sequence No.:305 / Sequence No.:306; Sequence No.:307 / Sequence No.:308; Sequence No.:309 / Sequence No.:310; Sequence No.:311 / Sequence No.:312; Sequence No.:315 / Sequence No.:316; Sequence No.:317 / Sequence No.:318; Sequence No. 319 / Sequence No. 320; and, Sequence No. 321 / Sequence No. 322.;

[0122] Although desirable, the present disclosure is not limited to each pair as indicated by itself. The group of VL and VH region species disclosed herein may be used to form distinct pairs, that is, various Fabs selected from the group of VL and VH region species provided.

[0123] Anti-CD47 VL is sequence number:1, sequence number:3, sequence number:5, sequence number:7, sequence number:9, sequence number:11, sequence number:13, sequence number:15, sequence number:17, sequence number:19, sequence number:21, sequence number:23, sequence number:25, sequence number:27, sequence number:29, sequence number:31, sequence number:33, sequence number:35, sequence number:37, sequence number:39, sequence number:41, sequence number:43, sequence number:45, sequence number:47, sequence number:49, sequence number:51, sequence number:53, sequence number:55, sequence number:57, sequence number:59, sequence number:61, sequence number:63, sequence number:65, sequence number:67, sequence number:69, sequence number:71, sequence number:73, sequence number:75, sequence number:77, sequence number:79, Sequence No.: 81, Sequence No.: 83, Sequence No.: 85, Sequence No.: 87, Sequence No.: 89, Sequence No.: 91, Sequence No.: 93, Sequence No.: 95, Sequence No.: 97, Sequence No.: 99, Sequence No.: 101, Sequence No.: 103, Sequence No.: 105, Sequence No.: 107, Sequence No.: 109, Sequence No.: 111, Sequence No.: 113, Sequence No.: 115, Sequence No.: 117, Sequence No.: 119, Sequence No.: 121, Sequence No.: 123, Sequence No.: 125, Sequence No.: 127, Sequence No.: 129, Sequence No.: 131, Sequence No.: 133, Sequence No.: 135, Sequence No.: 137, Sequence No.: 139, Sequence No.: 141, Sequence No.: 143, Sequence No.: 145, Sequence No.: 147, Sequence No.: 149, Sequence No.: 151, Sequence No.: 153, Sequence No.: 155, Sequence No.: 157, Sequence No.: 159, Sequence No.: 161, Sequence No.: 163, Sequence No.: 165, Sequence No.: 167, Sequence No.: 169, Sequence No.: 171, Sequence No.: 173, Sequence No.: 175, Sequence No.: 177, Sequence No.: 179, Sequence No.: 181, Sequence No.: 183, Sequence No.: 185, Sequence No.: 187, Sequence No.: 189, Sequence No.: 191, Sequence No.: 193, Sequence No.: 195, Sequence No.: 197, Sequence No.: 199, Sequence No.: 201, Sequence No.: 203, Sequence No.: 205, Sequence No.: 207,Sequence No.: 209, Sequence No.: 211, Sequence No.: 213, Sequence No.: 215, Sequence No.: 217, Sequence No.: 219, Sequence No.: 221, Sequence No.: 223, Sequence No.: 225, Sequence No.: 227, Sequence No.: 229, Sequence No.: 231, Sequence No.: 233, Sequence No.: 235, Sequence No.: 237, Sequence No.: 239, Sequence No.: 241, Sequence No.: 243, Sequence No.: 245, Sequence No.: 247, Sequence No.: 249, Sequence No.: 251, Sequence No.: 253, Sequence No.: 255, Sequence No.: 257, Sequence No.: 259, Sequence No.: 261, Sequence No.: 263, Sequence No.: 265, Sequence No.: 267, Sequence No.: 269, Sequence No.: 271, Sequence No.: 273, Sequence No.: 275, Sequence No.: 277, Selected from the group consisting of sequence number: 279, sequence number: 281, sequence number: 283, sequence number: 285, sequence number: 287, sequence number: 289, sequence number: 291, sequence number: 293, sequence number: 295, sequence number: 297, sequence number: 299, sequence number: 301, sequence number: 303, sequence number: 305, sequence number: 307, sequence number: 309, sequence number: 311, sequence number: 313, sequence number: 315, sequence number: 317, sequence number: 319, and sequence number: 321; Anti-CD47 VH is sequence number:2, sequence number:4, sequence number:6, sequence number:8, sequence number:10, sequence number:12, sequence number:14, sequence number:16, sequence number:18, sequence number:20, sequence number:22, sequence number:24, sequence number:26, sequence number:28, ​​sequence number:30, sequence number:32, sequence number:34, sequence number:36, sequence number:38, sequence number:40, sequence number:42, sequence number:44, sequence number:46, sequence number:48, sequence number:50, sequence number:52, sequence number:54, sequence number:56, sequence number:58, sequence number:60, sequence number:62, sequence number:64, sequence number:66, sequence number:68, sequence number:70, sequence number:72, sequence number:74, sequence number:76, sequence number:78, sequence number:80, Sequence No.: 82, Sequence No.: 84, Sequence No.: 86, Sequence No.: 88, Sequence No.: 90,Sequence No.: 92, Sequence No.: 94, Sequence No.: 96, Sequence No.: 98, Sequence No.: 100, Sequence No.: 102, Sequence No.: 104, Sequence No.: 106, Sequence No.: 108, Sequence No.: 110, Sequence No.: 112, Sequence No.: 114, Sequence No.: 116, Sequence No.: 118, Sequence No.: 120, Sequence No.: 122, Sequence No.: 124, Sequence No.: 126, Sequence No.: 128, Sequence No.: 130, Sequence No.: 132, Sequence No.: 134, Sequence No.: 136, Sequence No.: 138, Sequence No.: 140, Sequence No.: 142, Sequence No.: 144, Sequence No.: 146, Sequence No.: 148, Sequence No.: 150, Sequence No.: 152, Sequence No.: 154, Sequence No.: 156, Sequence No.: 158, Sequence No.: 160, Sequence No.: 162, Sequence No.: 164, Sequence No.: 166, Sequence No.: 168, Sequence No.: 170, Sequence No.: 172, Sequence No.: 174, Sequence No.: 176, Sequence No.: 178, Sequence No.: 180, Sequence No.: 182, Sequence No.: 184, Sequence No.: 186, Sequence No.: 188, Sequence No.: 190, Sequence No.: 192, Sequence No.: 194, Sequence No.: 196, Sequence No.: 198, Sequence No.: 200, Sequence No.: 202, Sequence No.: 204, Sequence No.: 206, Sequence No.: 208, Sequence No.: 210, Sequence No.: 212, Sequence No.: 214, Sequence No.: 216, Sequence No.: 218, Sequence No.: 220, Sequence No.: 222, Sequence No.: 224, Sequence No.: 226, Sequence No.: 228, Sequence No.: 230, Sequence No.: 232, Sequence No.: 234, Sequence No.: 236, Sequence No.: 238, Sequence No.: 240, Sequence No.: 242, Sequence No.: 244, Sequence No.: 246, Sequence No.: 248, Sequence No.: 250, Sequence No.: 252, Sequence No.: 254, Sequence No.: 255, Sequence No.: 258, Sequence No.: 260, Sequence No.: 262, Sequence No.: 264, Sequence No.: 266, Sequence No.: 268, Sequence No.: 270, Sequence No.: 272, Sequence No.: 274, Sequence No.: 276, Sequence No.: 278, Sequence No.: 280, Sequence No.: 282, Sequence No.: 284, Sequence No.: 286, Sequence No.: 288, Sequence No.: 290,An exemplary antibody otherwise functionally described herein is provided, selected from the group consisting of SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, and SEQ ID NO: 322.

[0124] Characteristics of the CD47xCD20 bispecific individual of the present invention

[0125] In vitro affinity measurements for the extracellular domain of the effector antigen CD47 demonstrated a 100–200-fold decrease in affinity for these variants initially. In vitro affinity measurements for the extracellular domain of CD47 demonstrated a 100–500-fold decrease in affinity for the variants. In vivo and in vitro cell-based studies on these unmodulated IgG1 1+1 heteromeric bispecific individuals confirmed effector-based apoptosis and reduced binding to non-target cell types compared to monospecific antibodies. Anti-CD20 VL: SEQ ID NO: 323; Anti-CD20 VH: SEQ ID NO: 324. The anti-CD20 LC constant region is SEQ ID NO: 343. The anti-CD20 HC constant region is SEQ ID NO: 345.

[0126] The bispecific entities described, illustrated, and claimed herein demonstrate, for example, selective binding to CD20-expressing cells, wherein the interaction between CD47 and the macrophage checkpoint inhibitor signal-regulatory protein alpha (SIRPα) is blocked. This increased selectivity for a monospecific anti-CD47 approach allows for the use of IgG1 Fc, which engages the activated fragment crystallizable gamma receptor (FcγR) to fully enable macrophages to engulf and destroy CD20-positive cells. Compared to the anti-CD20 antibody rituximab, for example, the anti-CD47 / anti-CD20 bispecific antibodies described and illustrated herein are more potent in inducing phagocytosis and ADCC in vitro.

[0127] In vitro cell-based studies demonstrate that the unmodulated CD47 bispecific individuals described herein activate antibody-dependent phagocytosis, complement-dependent cytotoxicity (CDC), and antibody-dependent cytotoxicity (ADCC). See Figs. 4a-4c and Figs. 5a-5c. Furthermore, pharmacokinetic (PK) and exploratory toxicity (E-tox) studies in cynomones demonstrate that the unmodulated CD47 bispecific individuals effectively deplete B cells and exhibit reduced binding to cynomones erythrocytes (RBCs) compared to parental monospecific anti-CD47 antibodies, thereby substantially confirming the success and medical value of the target-cell selective strategy described and claimed herein. The species exemplified herein exhibit favorable pharmacokinetics and CD20 while showing minimal adverse effects on hematological parameters following multiple administrations to non-human primates. + It proves the depletion of B cells.

[0128] In a specific embodiment, the CD47xCD20 bispecific entities provided herein designated as TPP-1360, TPP-1361, TPP-1367, and TPP-1362 comprise the following heavy chain and light chain sequences: TPP-1360 comprises (CD47 LC SEQ NO: 335; HC SEQ NO: 336) X (CD20 LC SEQ NO: 331; HC SEQ NO: 332). TPP-1361 comprises (CD47 LC SEQ NO: 333; HC SEQ NO: 334) X (CD20 LC SEQ NO: 331; HC SEQ NO: 332). TPP-1367 comprises (CD47 LC SEQ NO: 337; HC SEQ NO: 338) X (CD20 LC SEQ NO: 331; HC SEQ NO: 332). TPP-1362 contains (CD47 LC sequence number: 385; HC sequence number: 386) X (CD20 LC sequence number: 331; HC sequence number: 332).

[0129] With respect to TPP-1362, CD47 VL includes SEQ ID NO: 383. TPP-1362 CD47 VH includes SEQ ID NO: 384. TPP-1362 CD47 VL CDR includes SEQ ID NO: 377 (CDRL1); SEQ ID NO: 378 (CDRL2); and SEQ ID NO: 379 (CDRL3). TPP-1362 CD47 VH CDR includes SEQ ID NO: 380 (CDRH1); SEQ ID NO: 381 (CDRH2); and SEQ ID NO: 382 (CDRH3).

[0130] An exemplary series of bispecific specimens within the genus otherwise described herein is proven to exhibit pharmacological properties that generally indicate the therapeutic value of the genus.

[0131] For example, these highly evaluated species exhibit high affinity for CD20 and uncontextualized affinity for CD47, effective CD47 blockade, cyno-cross-reactivity, excellent physicochemical properties (solubility, stability, expression), and low immunogenicity prediction (EpiVax). See Example 13 and Fig. 18. IgG1 heterodimeric forms and Fc provide reliable production in sufficient volume and purity using a standard CHO process, along with appropriate potency, yield, production quality, and liquid formulation in a stepwise manner. These exemplary highly evaluated species demonstrate superior CD20 compared to CC-90002. + It exhibits the in vitro phagocytic ability of tumor cells and ADCC more potent than rituximab. These exemplary highly evaluated species also show a significant reduction in cyno B cells in peripheral blood and lymphoid tissues. These exemplary highly evaluated species also CD20 - CD47 + It exhibits a minimal sink effect without binding to healthy cells (RBCs and platelets). These exemplary, highly evaluated species also exhibit acceptable PK parameters that support, for example, weekly dosing.

[0132] For example, the RBC binding ability of TPP-1360 was extensively evaluated in purified human RBCs and in co-cultures of human RBCs and tumor cells. As shown in Fig. 8, TPP-1360 is CD47 + / CD20 + It selectively binds to Raji cells but CD47 + / CD20 - It is proven that it does not bind to human RBCs. Furthermore, in the co-culture of Raji cells and human RBCs, TPP-1360 is CD47 + / CD20 +It exhibited dose-dependent binding to Raji cells but did not bind to human RBCs even at high concentrations of 1 mg / mL. See Fig. 9. Conversely, the CD47 wild-type / CD20 bispecific TPP-2 significantly bound to both Raji cells and human RBCs. Additionally, TPP-1360 does not exhibit binding to purified cyno RBCs from multiple donors.

[0133] TPP-1360, an exemplary species bispecific agent of the present disclosure, is the first antibody to co-target CD47 and CD20, designed to bind to CD20 with high affinity and optionally CD47 with uncoordinated affinity. When bound to CD20-expressing cells, for example, TPP-1360 not only blocks the interaction between CD47 and the macrophage checkpoint inhibitor SIRPα but also engages in the activation of FcγR, thereby fully enabling macrophages to engulf and destroy CD20-positive cells. Robust in vitro activity is induced, for example, by TPP-1360 to eliminate cancer cells through various modes of action, including phagocytosis, ADCC, and CDC. TPP-1360, an example of the bispecific agent described herein, provides enhanced pharmacological activity compared to rituximab and CC-90002.

[0134] The CD47xCD20 bispecific entities described and claimed herein demonstrate enhanced phagocytosis as a single agent compared to rituximab or CC-90002. The phagocytic activity of the CD47xCD20 bispecific entities described herein generally correlates with their CD47 binding affinity. The single agent activity of the CD47xCD20 bispecific entities described herein is equivalent to the combination of CC-90002 and rituximab in inducing phagocytosis.

[0135] The CD47XCD20 bispecific entity described herein demonstrates improved ADCC in rituximab-sensitive and resistant tumor cells compared to single-acting anti-CD47 activity.

[0136] The CD47XCD20 bispecific individual described herein demonstrates superior efficacy to rituximab in vivo in the Raji NOD-SCID model. See Example 15.

[0137] TPP-1360 enhances both phagocytosis and ADCC activity compared to rituximab. Furthermore, TPP-1360 and the related bispecific entities described, illustrated, and claimed herein possess different modes of action, including phagocytosis, ADCC, and CDC, compared to T cell activation; thus, they are now distinguished in clinical practice from CD20xCD3 bispecific T-cell engaging antibodies such as Regeneron’s REGN1979 or Roche’s mosunetuzumab. Additionally, the toxicity profile differs from that of CD20xCD3 (potential hematological toxicity versus cytokine release syndrome). In particular, T-cell engaging antibodies are potent immune engaging antibodies capable of triggering apoptosis in non-target cells expressing very low levels of the target antigen; therefore, the target antigen must be extremely specific, or the anti-targeting arm of the bispecific individual must be tuned to utilize masking techniques or to distinguish between the expression levels of the target antigen in normal and diseased tissues. Accordingly, the anti-CD3 portion of the bispecific individual must be precisely tuned to prevent cytokine release due to systemic activation of T-cells. Importantly, as a representative example of the present invention, TPP-1360 demonstrates favorable elimination kinetics while showing minimal adverse effects on hematological parameters after multiple administrations to non-human primates. The CD47xCD20 bispecific antibody of the present invention is being developed as an intravenous (IV) injectable therapy, particularly for the treatment of patients with B-lymphoma who are refractory and / or resistant to current therapies.

[0138] The bispecific entities described herein are provided for a method of treating and / or controlling tumors, tumor cells, and cancers, including but not limited to cells undergoing abnormal proliferation, hematological oncological conditions, hematological malignancies, lymphoproliferative disorders, B-cell disorders, B-cell malignancies, and / or B-cell lymphomas. The bispecific entities of the present invention are formulated and administered in accordance with the current state of technology of antibodies as therapeutic entities. For example, standards for the formulation and administration of IgG1 antibodies are widely known in the art. The antibodies described herein are administered, for example, as an intravenous (IV) injectable treatment for patients with CD20-positive B-cell lymphoma. The present invention relates to a method for controlling tumor cells, comprising the step of administering an effective amount of the bispecific entities described herein to a patient in need thereof. Tumor cells refer to cancer cells, including but not limited to cells undergoing abnormal proliferation, hematological oncological conditions, hematological malignancies, lymphoproliferative disorders, B-cell disorders, B-cell malignancies, and B-cell lymphomas.

[0139] The CD47XCD20 bispecific entity described herein is provided for use in a method for treating B-cell disorders or B-cell malignancies, comprising the step of administering an effective amount of the bispecific entity described herein to a patient in need thereof.

[0140] Relative growth and human pharmacokinetics are evaluated for the bispecific subjects described herein. The subjects of the present invention include an unadjusted CD47 binding arm and a regular CD20 (rituximab) binding arm. Considering the unadjusted binding affinity of the CD47 arm, the target-mediated drug batch (TMDD) for the bispecific subjects is potentially driven primarily by CD20 binding. Therefore, in certain embodiments, the clinical dose will be in the range currently used for rituximab. Based on doses of 10, 20, and 100 mg / kg in cynomolgus monkeys, the terminal half-life is approximately 7 days. The CD20-mediated TMDD is based on additional preclinical and clinical data for rituximab. The first clinical study in humans is an open-label, multicenter Phase 1 / 1b study to evaluate safety and tolerability in subjects with relapsed or refractory CD20+ NHL who have undergone rituximab and / or other CD20-targeted therapies. The stepwise increase in dose starts at less than 1 mg / kg and then increases stepwise to 10 mg / kg, which is the current clinical dose for rituximab. Synomolgus monkeys were administered 20 mg / kg twice on day 1 and day 15. The bispecific agent described herein was found to be well tolerated as a monoagonist in the NHP eTOX study based on dose-proportional exposure. The mammalian or human dose of the bispecific agent described herein is within the range of about 3 mg / kg to about 20 mg / kg. Additional mammalian or human doses of the bispecific agent described herein are particularly within the range of about 5 mg / kg to about 15 mg / kg. The mammalian or human dose of the bispecific agent described herein is also about 7 mg / kg to about 13 mg / kg. The dosage regimen of the bispecific agent described herein is once every about 5 days, or once every about 1 week (7 days), or once every about 10 days, or once every about 2 weeks.

[0141] The manufacturing process of the bispecific antibodies described herein may follow a typical Chinese hamster ovary (CHO) manufacturing platform. A common contaminant observed during the purification of these bispecific antibodies is the half-antibody, and a specific purification protocol is required to remove it. After the expression of the 4-chain bispecific antibody in Chinese hamster ovary cells, Protein A is used as the first step for purifying the IgG-based bispecific antibody. After this first step, typically two species exist: the desired 4-chain bispecific antibody and the half-antibody. In most cases, ion exchange chromatography is sufficient to separate these two species, but hydrophobic interaction chromatography may be required for other species. The correct pairing of the LC must be evaluated by mass spectrometry, and misassembled impurities must be removed by additional protein purification methods such as ion exchange or hydrophobic interaction chromatography. According to the secondary purification approach, morphological homogeneity can be refined and ensured using manufacturing size exclusion chromatography (SEC) while the buffer exchanges four-chain bispecific entities. Final quality control should include analytical SEC, mass spectrometry, and in vitro binding evaluation using different antigens to ensure the morphological and chemical integrity of the bispecific entities. See, for example, JB Ridgway et al., Protein Eng. 9 (1996) 617-621; K. Gunasekaran et al., J. Biol. Chem. 285 (2010) 19637-19646. The preferred monomeric elements of the IgG1 1+1 heteromers described herein each contain specific LC and HC invariant regions discussed above and identified herein to reduce the tendency toward homomerization during the production of the IgG1 1+1 heteromer format.

[0142] Examples

[0143] Example 1: Non-adjustment of CC-90002

[0144] A reasonable design to reduce the affinity of the uncoordinated parent version of the CC-90002(408_437) anti-CD47 arm was made possible by the crystal structure of an anti-CD47 Fab bound to the extracellular domain of CD47. The epitope bound by CC-90002 is identical to the epitope of the original murine anti-CD47 2A1 bound to human CD47. See U.S. Patent No. 9,045,541.

[0145] The variable domain of 2A1 was humanized, and the final antibody was named "QN," consisting of HC_2.3Q and LC_N, which was ultimately developed into the IgG4 P / E format (CC-90002). QN was further modified by introducing residues into the variable heavy chain domain for improved cell-free expression, and this HC variant was named "HC_Q_5_MUT". HC_Q_5_MUT HC and LC_N were further modified to reduce immunogenicity using in silico modeling and in silico prediction of immunogenicity, and these were collectively referred to as "CD47 2.0". For improved pharmacokinetics, additional variants were designed in the variable heavy chain and variable light chain domains of CD47 2.0 LC_1147_2 and CD47 2.0 HC_434, and these were referred to as "CD47 3.0". WO2016109415 (US.20170369572); WO2018009499 (US.20190241654); and WO2018183182, each of which is incorporated herein by reference.

[0146] The anti-CD47 epitope covers a large surface area, and residues from both the light chain (LC) and the heavy chain (HC) participate in the interaction.

[0147] To reduce the affinity of anti-CD47 arm to CD47, CD47 interacting residues from both LC and HC were applied to in silico mutagenesis using the "residue scan" module of a molecular operating environment (MOE) modeling program. This process generated a library of thousands of variants with a wide range of predicted affinities. Each in silico Fab variant was modeled to calculate the predicted change in stability (dStability) or change in affinity to CD47 ECD (dAffinity). Over 5,000 variants with positive dAffinity scores (predicted to have lower affinity compared to the parent Fab) and negative dStability scores (predicted to have higher stability compared to the parent Fab) were analyzed using immunogenicity assessment software to identify variants predicted to have low immunogenicity. Among these, 143 low immunogenicity risk Fab variants with predicted Kd for CD47 in the range of 10 nM to 1 mM were selected for cell-based testing.

[0148] To screen target-cell selective anti-CD47 Fab, selected anti-CD47 Fab variants were constructed as IgG1 fusions and paired with anti-EGFR arms from cetuximab. The proper assembly of the four-chain bispecific entities was made possible by the presence of the Fab and Fc substitutions described herein in all four chains. Four-chain bispecific entities containing 143 selected variants were transiently expressed in Expi-CHO cells, and the bispecific entities were purified in a single step using autoprotein A beads. To identify target-cell selective bispecific entities, the variants were tested in two experiments. The first experiment measured the ability of unmodulated anti-CD47 x anti-EGFR bispecific entities to bind to non-target Raji cell lines that express CD47 antigen but not EGFR antigen. The second experiment measured the ability of unmodulated anti-CD47 x anti-EGFR bispecific variants to block SIRPα binding to target Fadu cell lines expressing CD47 antigen and EGFR antigen. These experiments yielded a set of eight variants that exhibited a 10 to 20-fold reduced affinity to non-target CD47+ / EGFR- Raji cell lines compared to the unmodulated anti-CD47x anti-EGFR parental antibody, but were still able to block 75-90% of SIRPα binding to CD47+ / EGFR+ Fadu target cell lines.

[0149] Rituximab anti-CD20 arm was paired with eight similarly unmodulated anti-CD47 variants using IgG1 Fc. The unmodulated CD47xCD20 bispecific antibody was observed to have reduced binding to CD47+ / CD20- non-target Fadu cell lines compared to the unmodulated CD47xCD20 parent antibody, but was still able to block 75-90% of SIRPα binding to CD47 and CD20-positive target Raji cell lines.

[0150] The evaluation of the potential for further development of variants led to the selection of VH E59Y / S102E, a single -CD47 Fab variant cloned into three CC-90002-derived frameworks, namely TPP-1367, TPP-1360, and TPP-1361, for toxicology testing in synomolgus monkeys.

[0151] Example 2: Summary of SPR binding results for the bispecific entities described herein

[0152] The affinity of TPP-1360 and TPP-1362 for CD47 was measured using surface plasmon resonance (SPR) experiments. These two antibodies were tested for binding to human CD47 and synomolgus CD47 and were found not to bind to mouse CD47. TPP-1360 was measured to have an affinity of 1.7 μM Kd for human CD47 ECD, which reflects a ~350-fold decrease in affinity compared to the parent anti-CD47 binder. The affinity of TPP-1360 for synomolgus CD47 ECD was found to be 4.51 μM Kd. TPP-1362 was measured to have an affinity of 0.796 μM Kd for human CD47 ECD, reflecting a ~150-fold decrease in affinity compared to the parent anti-CD47 binder. The affinity of TPP-1362 for synomolgus CD47 ECD was found to be 2.06 μM Kd. Finally, in addition to the measured affinities, the sandwich SPR assay demonstrated that both bind simultaneously to CD47 and CD20.

[0153] Example 3: Dose-response of binding and SIRPα blockade of an exemplary bispecific entity

[0154] Dose-response curves were generated for the blockade of TPP-1360 and TPP-1362 by human SIRPα binding to various CD20s in non-Hodgkin lymphoma tumor cell lines. Cell lines were co-cultured with increasing concentrations of the bispecific agent, followed by the addition of human SIRPα to saturation concentrations. In addition to the bispecific agent, rituximab and a parental anti-CD47 binder (TPP-23, 408_437 containing IgG1) were included for reference. Cells were washed and then incubated with secondary antibodies to measure the amount of SIRPα bound to the tumor cells. For the cell line OCI-Ly3 (DLBCL cell line), TPP-1360 was found to have an EC50 of 1.30 nM, and TPP-1362 was found to have an EC50 of 0.70 nM. In the case of the Raji cell line (B-lymphocyte Burkitt lymphoma cell line), TPP-1360 was found to have an EC50 of 1.64 nM, and TPP-1362 was found to have an EC50 of 1.10 nM. The parent anti-CD47, TPP-23, had an IC50 of 0.11 nM to block human SIRPα binding to OCI-Ly3 cells, as shown in Fig. 21. 50 It was found to have. Rituximab did not affect SIRPα binding.

[0155] Example 4: Dose-response to phagocytosis

[0156] Dose-response curves were generated for the activation of TPP-1360 and TPP-1362 of phagocytosis against various CD20-expressing non-Hodgkin lymphoma tumor cell lines. Human monocytes were differentiated into macrophages and then added to the co-cultured tumor cell lines at increasing concentrations of bispecific individuals. In addition to bispecific individuals, rituximab and a parental anti-CD47 binder (TPP-23) were included for reference. The number of phagocytic events was measured using image-based quantification methods based on the fluorescent labeling of macrophages and tumor cells. For the OCI-Ly3 cell line, TPP-1360 was found to have an IC50 of 1.4 nM, and TPP-1362 was found to have an IC50 of 0.43 nM. In the case of the Raji cell line, TPP-1360 was found to have an IC50 of 1.8 nM and TPP-1362 was found to have an IC50 of 0.37 nM.

[0157] Example 5: Combined study using human and cyno RBC and hemagglutination tests

[0158] The binding of specific bispecific individuals to human and cynomolgus monkey RBCs was determined to evaluate their non-target cell binding potential. RBCs were isolated from whole blood and co-cultured with increasing concentrations of the exemplary bispecific individuals. Binding was expressed as a percentage of the binding amount observed at 2 μg / ml of the parental anti-CD47 binder (TPP-23). ​​At 200 μg / ml, TPP-1360 and TPP-1361 bound to < 1% of the parental anti-CD47 binding observed to human RBCs. Similarly, at 200 μg / ml, TPP-1360 bound to < 1% of the parental anti-CD47 binding observed to cynomolgus RBCs. At 200 μg / ml, a higher degree of binding was observed for TPP-1362 to synomolgus RBCs, representing 2–3% binding of the parent anti-CD47 binder. Finally, the parent anti-CD47 binder to the two leads demonstrated no hemagglutination of human RBCs at 200 μg / ml. Similarly, both TPP-1360 and TPP-1361 showed no hemagglutination at 200 μg / ml. BRIC6, a known hemagglutination antibody, was used as a positive control.

[0159] Example 6: Binding study of human PBMC and whole blood

[0160] The binding of the bispecific populations described herein to human peripheral blood mononuclear cells (PBMCs) was evaluated. Compared to the parent anti-CD47 binders TPP-23 and rituximab, the TPP-1360 bispecific population showed less binding to all cell types except B-cells, which showed significant binding through the presence of the anti-CD20 Fab moiety.

[0161] Example 7: First round lead synomolgus PK

[0162] Synomolgus PK experiments were performed on the exemplary bispecific populations described herein. Synomolgus monkeys were administered 20 mg / kg twice on days 1 and 15. B-cell depletion was observed. From these studies, TPP-1360 and TPP-1362 were selected for further study in the Synomolgus monkey exploratory toxicology (E-tox) study, as described in Example 8.

[0163] Example 8: Second round lead synomolgus E-tox

[0164] Synomolgus E-tox experiments were performed with TPP-1360 and TPP-1362. For TPP-1360, Synomolgus monkeys were administered 100, 20, and 10 mg / kg once weekly for 2 weeks, followed by a 2-week non-treatment period. A second TPP-1360 sub-arm was tested at 10 mg / kg twice weekly for 2 weeks, followed by a 2-week non-treatment period. For TPP-1362, Synomolgus monkeys were administered 60, 20, and 10 mg / kg once weekly for 2 weeks, followed by a 2-week non-treatment period. After testing a second TPP-1362 agent at 10 mg / kg twice a week for 2 weeks, a 2-week non-dose period was also followed by administration at 20 mg / kg twice on days 1 and 15. This study indicated that TPP-1360 is widely tolerated, demonstrates deep B-cell depletion, and achieves dose-proportional exposure, confirming that it avoids sink and is therefore target-cell selective.

[0165] Example 9: In vitro pharmacology

[0166] A. Human whole blood combination

[0167] To evaluate the specificity of TPP-1360, its binding profile was first assessed in whole blood using flow cytometry. Across two donors, 200 nM TPP-1360 demonstrated selective binding to B cells in human whole blood, exhibiting minimal or no binding to platelets or red blood cells, substantially shifting binding signals to B cells, and somewhat weakly shifting to T cells, monocytes, and NK cells. See Fig. 7.

[0168] Figure 7 shows, for example, that the bispecific TPP-1360 binds primarily to B cells, with very small amounts of binding to the other listed cell types, possibly due to higher levels of CD47 found in blood cells or the contribution of Fc involved in the Fc receptor expressed on NK cells and monocytes. Conversely, TPP-23, a high-affinity CD47 monospecific antibody, binds to all of these cell types due to the universal expression of CD47 and the high affinity for CD47 found in TPP-23.

[0169] The overall binding profile of TPP-1360 in human whole blood is similar to that of rituximab. Conversely, TPP-23, a parental CD47 mAb used as a control for CD47 expression, significantly bound to all cell populations in human blood.

[0170] B. Tumor cell binding

[0171] In addition, for example, the RBC binding ability of TPP-1360 was extensively evaluated in purified human RBCs and in co-cultures of human RBCs and tumor cells. As shown in Fig. 8, TPP-1360 is CD47 + / CD20 + It selectively bound to Raji cells but CD47 + / CD20 -It did not bind to human RBCs. Furthermore, in the co-culture of Raji cells and human RBCs, TPP-1360 CD47 + / CD20 + It exhibited dose-dependent binding to Raji cells but did not bind to human RBCs even at high concentrations of 1 mg / mL. See Fig. 9. Conversely, TPP-2, a parental CD47 type / CD20 bispecific individual, significantly bound to both Raji cells and human RBCs. Additionally, TPP-1360 does not exhibit binding to purified cyno RBCs from multiple donors.

[0172] C. SIRPα competition

[0173] CD20 + / CD47 + After demonstrating selective binding to cells, the ability of TPP-1360 to antagonize the interaction between cell surface CD47 and human SIRPα was evaluated using an in vitro competitive assay. TPP-1360 had mean EC50 values ​​of 1.30 nM and 1.64 nM, respectively, and CD20 + / CD47 + Recombinant human SIRPα-Fc binding to human CD47 expressed on the surface of lymphoma cell lines OCI-Ly3 and Raji was potently blocked. See Figs. 10 and 11. Fig. 10 is For example, TPP-1360 is CD20 + / CD47 + It illustrates the fact that the binding of recombinant human SIRPα-Fc to human CD47 expressed on the surface of lymphoma cell line OCI-Ly3 was potently and completely blocked. Fig. 11 For example, TPP-1360 is CD20 + / CD47 +It demonstrates that recombinant human SIRPα-Fc binding to human CD47 expressed on the surface of the lymphoma cell line Raji was potently and completely blocked. Conversely, rituximab or the control bispecific antibody TPP-1480 (anti-CD20 / hen-al-lysozyme) could not compete with human SIRPα-Fc binding to the same cell line. The data presented herein also demonstrate that the efficacy of TPP-1360 in blocking human SIRPα-CD47 interactions is lower than that of TPP-23, which is consistent with the attenuated affinity of TPP-1360 for human CD47.

[0174] Example 10: Functional Activity: Human Macrophage Phagocytosis

[0175] This example involves "eaten" CD20 inside labeled macrophages + CD47 + As determined in vitro by the automation coefficient of tumor cells, it demonstrates the ability of TPP-1360 to trigger tumor phagocytosis.

[0176] The expression of CD20 and CD47 was first confirmed in each target tumor cell line (OCI-Ly3, Raji, REC-1, and RIVA) by quantifying antibody binding ability (ABC) using flow cytometry (Denny TN et al., Cytometry. 1996 Dec;26(4):265-74). All four cell lines express high levels of CD47 and CD20. Table 1.

[0177] Table 1: Expression of CD47 and CD20 antigens on lymphoma cell surfaces

[0178]

[0179] Next, the titrated antibody was added to pre-differentiated macrophages and co-cultured with carboxyfluorescein succinimidyl ester (CSFE)-labeled tumor cells opsonized with TPP-1360. Phagocytic activity was quantitatively determined by the number of labeled tumor cells within the labeled macrophages. Green intensity (CFSE) was measured in each CD14 allophycocyanin (APC)-labeled macrophage, and CFSE-positive macrophages were identified using a threshold gate. A threshold of approximately 1,000 MFI (mean fluorescence intensity) was observed across the experiment with a deviation of less than several hundred MFI. For each sample, the calculated percentage of phagocytosis was determined as follows: [(Number of CFSE-positive macrophages) / (Total number of macrophages)] x 100. Across at least two donors, TPP-1360 treatment resulted in 4 CD20 + Macrophage-mediated phagocytosis was induced in malignant B cell lines. Representative data from one donor are presented in Figures 12 (Raji cells), 13 (OCI-Ly3 cells), 14 (REC-1 cells), and 15 (RIVA cells). After calculating the area under the curve, a paired t-test was performed to determine the statistical significance of TPP-1360 compared to rituximab. See Figure 16. The data demonstrate that TPP-1360 treatment triggered significantly more efficient phagocytosis than rituximab in Raji and OCI-Ly3 cells, which may be attributed to the simultaneous blockade of SIRPα-CD47 interactions and the involvement of activating receptors such as FcγR by TPP-1360.

[0180] Example 11: Pharmacokinetics

[0181] To determine the pharmacokinetic (PK) profiles of the bispecific individuals (antibody species, TPP-1360, TPP-1361, TPP-1362, and TPP-1367) described herein, non-GLP studies were performed in mice and cynomolgus monkeys. A single-dose mouse PK study was conducted using naive, female, NOD / SCID mice administered 10 mg / kg of the antibody via intraperitoneal (IP) injection. Rare PK sampling (n = 4 per time point) was performed over a 72-hour course, and all animals exhibited detectable antibody species concentrations throughout the study period. The calculated half-life was 3.4 days, but this may be underestimated considering the sampling period. To evaluate the PK profile in cynomolgus monkeys, a repeated-dose exploratory toxicology study was performed, and 20 mg / kg of the antibody species was administered to three naive male monkeys via IV bolus injection on days 1 and 15. The antibody species After repeated dosing, systemic exposure was achieved, and antibody species were detectable in the serum of 2 out of 3 monkeys throughout the study period (336 hours after 15 days of dosing). Additionally, samples were collected as part of the repeated-dose study for hematological and immunophenotypic evaluation. The observed depletion of B-lymphocytes demonstrates the drug's efficacy in vivo. Overall, antibody species exposure was maintained throughout the study period in both the single-dose mouse and repeated-dose monkey studies, and similar half-lives ranging from 3 to 3.5 days were reported between the two studies.

[0182] Example 12: Safety Profile

[0183] This exemplary series of highly evaluated species exhibits an acceptable toxicological profile and was well tolerated, for example, up to the highest tested dose of 100 mg / kg QW. Toxicological kinetics were evaluated as part of a 28-day exploratory toxicology study in cynomolgus monkeys. TPP-1360 was administered via IV injection to cynomolgus monkeys (4 per group) at a dose level of 10 mg / kg (BIW) on days 1, 4, 8, 11, and 15, or at 20 and 100 mg / kg (QW) on days 1, 8, and 15. Serum concentrations were measured by a sandwich ELISA using anti-rituximab antibody for capture and goat anti-human IgG Fc for detection. Following multiple IV doses of 10, 20, or 100 mg / kg, systemic exposure to TPP-1360 was achieved at all dose levels and maintained by all animals throughout the study period. TPP-1360 was C across the 20 and 100 mg / kg dose groups max and AUC 0-168 A linear TK with approximately dose-proportional increase was observed. After the first dose, clearance was similar across the 10–100 mg / kg dose range, suggesting target saturation at the 10 mg / kg dose. R AUCThe values ​​represent partial TPP-1360 accumulation by dose 5 and dose 3 in the 10 (BIW) and 100 (QW) mg / kg dose groups, respectively. The mean calculated half-life ranged from 2 to 4 days depending on the dose level and dose regimen. Anti-drug antibodies were detected in 5 out of 8 animals tested 15 days prior to administration and in 5 out of 6 animals tested on day 29 of the study. Anti-drug antibodies affected TPP-1360 exposure, as evidenced by the reduction observed in ADA-positive animals. TPP-1360 was well tolerated up to the highest tested dose of 100 mg / kg QW. A reduction in B cells in peripheral blood and multiple lymphoid tissues was observed at 10 mg / kg BIW and ≥ 20 mg / kg QW, demonstrating potent pharmacokinetic activity. Administering 10 mg / kg BIW did not provide additional benefit compared to 20 mg / kg QW. In addition to effects on B cells, TPP-1360 also reduced T cells and NK cells at all dose levels, neutrophils at ≥ 20 mg / kg QW, and erythrocytes at 100 mg / kg QW. However, no test-related reduction in platelets was observed. The reduction in T cells, NK cells, neutrophils, and erythrocytes is believed to be mediated by the CD47 arm of TPP-1360, as these cells do not express CD20.

[0184] Example 13: Immunogenicity

[0185] Interactive Screening and Protein Remodeling Interface (ISPRI) software developed by EpiVax is an in silico computer method used to evaluate potential antibody immunogenicity in humans and is known as a clinically widely established T cell-dependent analysis tool (Fig. 18). The VH and VL amino acid sequences of TPP-1360 were analyzed for putative T effectors and T regulatory hotspots and were found to have a low risk of immunogenicity.

[0186] Example 14: Raji xenograft model

[0187] The CD47XCD20 bispecific individual described herein demonstrates superior efficacy to rituximab in vivo in a Raji NOD-SCID model. The objective of this study was to determine the monotherapy antitumor activity of TPP-1360 or TPP-1362 in a Raji xenograft model expressing lower levels of CD20 and higher levels of CD47. Raji cells were inoculated into the right flank of female NOD-SCID mice. The tumor was approximately 270 mm 3 Dosage was initiated when the size was reached. TPP-1360 and TPP-1362 were tested at 10 and 30 mg / kg for 2 weeks with once-weekly (QW) dosing. Rituximab, which is divalent to CD20, was used as a comparator with the same dosing paradigm. The final tumor volume reduction was approximately 2000 mm³ in the isoplastic control (anti-RSV IgG1) group, with an average tumor volume of that size. 3The end point of the study, at which the TVR was reached, was determined on day 25. Significant (p < 0.0001) antitumor activity of TPP-1360 with a 52% reduction in tumor volume was observed at both 10 and 30 mg / kg, QW, and significant antitumor activity of TPP-1362 with a 62% reduction in tumor volume was observed at 10 mg / kg QW or 64% at 30 mg / kg QW. (Figs. 19-20) At the same dosing regimen, rituximab showed TVRs of 33% and 38% at 30 and 10 mg / kg, respectively. The antitumor activity of TPP-1360 at 30 mg / kg, QW was significantly (p < 0.01) better than that of rituximab at the corresponding dose level, suggesting a contribution of the CD47 subarc to the antitumor activity of TPP-1360. The antitumor activity of TPP-1362 at 30 mg / kg, QW was also significantly (p<0.0001) better than that of rituximab at the corresponding dose level, suggesting a contribution of the CD47 subarc to the antitumor activity of TPP-1362. There was no significant body weight loss in animals treated with isomorphic controls, TPP-1360, TPP-1362, or rituximab.

[0188] Sequence list

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] All publications and patents mentioned herein are incorporated by reference. Various variations and modifications of the subject matter described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to specific embodiments, it should be understood that the invention as claimed should not be excessively limited to such embodiments. In practice, various modifications to practice the invention are intended to be obvious to those skilled in the art and within the scope of the following claims.

Claims

Claim 1 A bispecific antibody in the form of an IgG1 1+1 heterodimer comprising at least one Fab portion binding to CD47 and at least one Fab portion binding to CD20, wherein the Fab portion binding to CD47 exhibits a low affinity for CD47 of 0.1 μM to 5.0 μM; the Fab portion binding to CD20 comprises light chain variable (VL) CDRs: SEQ ID NO: 353 (CDRL1), SEQ ID NO: 354 (CDRL2), and SEQ ID NO: 355 (CDRL3); and heavy chain variable (VH) CDRs: SEQ ID NO: 356 (CDRH1), SEQ ID NO: 357 (CDRH2), and SEQ ID NO: 358 (CDRH3); and the Fab portion binding to CD47 comprises (a) a VL region comprising VL CDRs: SEQ ID NO: 359 (CDRL1), SEQ ID NO: 360 (CDRL2), and SEQ ID NO: 361 (CDRL3); and VH region comprising VH CDR: SEQ ID NO: 362 (CDRH1), SEQ ID NO: 363 (CDRH2) and SEQ ID NO: 364 (CDRH3); (b) VL region comprising VL CDR: SEQ ID NO: 365 (CDRL1), SEQ ID NO: 366 (CDRL2) and SEQ ID NO: 367 (CDRL3); and VH region comprising VH CDR: SEQ ID NO: 368 (CDRH1), SEQ ID NO: 369 (CDRH2) and SEQ ID NO: 370 (CDRH3), or (c) VL region comprising VL CDR: SEQ ID NO: 371 (CDRL1), SEQ ID NO: 372 (CDRL2) and SEQ ID NO: 373 (CDRL3); and VH CDR: comprising a VH region including SEQ ID NO: 374 (CDRH1), SEQ ID NO: 375 (CDRH2) and SEQ ID NO: 376 (CDRH3); a bispecific antibody that selectively binds to CD47 in tumor cells and binds to less than 5% of CD47 in CD20- / CD47+ cells. Claim 2 A bispecific antibody that selectively binds to B cells in claim 1. Claim 3 In paragraph 2, a bispecific antibody comprising an anti-CD47 light chain (LC) constant region including SEQ ID NO:

340. Claim 4 In paragraph 3, a bispecific antibody comprising an anti-CD47 heavy chain (HC) constant region including SEQ ID NO:

342. Claim 5 In paragraph 2, a bispecific antibody that selectively binds to malignant B cells. Claim 6 In paragraph 4, a bispecific antibody that selectively binds to malignant B cells. Claim 7 In claim 6, a bispecific antibody comprising an anti-CD20 light chain (LC) constant region including SEQ ID NO:

344. Claim 8 A bispecific antibody according to claim 7, comprising an anti-CD20 heavy chain (HC) constant region including SEQ ID NO:

346. Claim 9 A bispecific antibody according to claim 2, wherein the Fab portion binding to CD47 comprises (i) a light chain variable (VL) region selected from the group consisting of SEQ ID NO: 317, SEQ ID NO: 319, and SEQ ID NO: 321; and (ii) a heavy chain variable (VH) region selected from the group consisting of SEQ ID NO: 318, SEQ ID NO: 320, and SEQ ID NO:

322. Claim 10 A bispecific antibody according to claim 8, wherein the Fab portion binding to CD47 comprises (i) a light chain variable (VL) region selected from the group consisting of SEQ ID NO: 317, SEQ ID NO: 319 and SEQ ID NO: 321; and (ii) a heavy chain variable (VH) region selected from the group consisting of SEQ ID NO: 318, SEQ ID NO: 320 and SEQ ID NO:

322. Claim 11 A bispecific antibody according to claim 2, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 359 (CDRL1), SEQ ID NO: 360 (CDRL2), and SEQ ID NO: 361 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 362 (CDRH1), SEQ ID NO: 363 (CDRH2), and SEQ ID NO: 364 (CDRH3). Claim 12 A bispecific antibody according to claim 2, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 365 (CDRL1), SEQ ID NO: 366 (CDRL2), and SEQ ID NO: 367 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 368 (CDRH1), SEQ ID NO: 369 (CDRH2), and SEQ ID NO: 370 (CDRH3). Claim 13 A bispecific antibody according to claim 2, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 371 (CDRL1), SEQ ID NO: 372 (CDRL2), and SEQ ID NO: 373 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 374 (CDRH1), SEQ ID NO: 375 (CDRH2), and SEQ ID NO: 376 (CDRH3). Claim 14 A bispecific antibody according to claim 8, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 359 (CDRL1), SEQ ID NO: 360 (CDRL2), and SEQ ID NO: 361 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 362 (CDRH1), SEQ ID NO: 363 (CDRH2), and SEQ ID NO: 364 (CDRH3). Claim 15 A bispecific antibody according to claim 8, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 365 (CDRL1), SEQ ID NO: 366 (CDRL2), and SEQ ID NO: 367 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 368 (CDRH1), SEQ ID NO: 369 (CDRH2), and SEQ ID NO: 370 (CDRH3). Claim 16 A bispecific antibody according to claim 8, wherein the Fab portion binding to CD47 comprises a light chain variable (VL) region including VL CDR: SEQ ID NO: 371 (CDRL1), SEQ ID NO: 372 (CDRL2), and SEQ ID NO: 373 (CDRL3); and a heavy chain variable (VH) region including VH CDR: SEQ ID NO: 374 (CDRH1), SEQ ID NO: 375 (CDRH2), and SEQ ID NO: 376 (CDRH3). Claim 17 A bispecific antibody according to claim 11, comprising a VL region containing SEQ ID NO: 317; and a VH region containing SEQ ID NO:

318. Claim 18 A bispecific antibody according to claim 12, comprising a VL region containing SEQ ID NO: 319; and a VH region containing SEQ ID NO:

320. Claim 19 A bispecific antibody according to claim 13, comprising a VL region containing SEQ ID NO: 321; and a VH region containing SEQ ID NO:

322. Claim 20 A bispecific antibody according to claim 14, comprising a VL region containing SEQ ID NO: 317; and a VH region containing SEQ ID NO:

318. Claim 21 A bispecific antibody according to claim 15, comprising a VL region containing SEQ ID NO: 319; and a VH region containing SEQ ID NO:

320. Claim 22 A bispecific antibody according to claim 16, comprising a VL region containing SEQ ID NO: 321; and a VH region containing SEQ ID NO:

322. Claim 23 A bispecific antibody according to claim 1, comprising an anti-CD47 VL region comprising SEQ ID NO: 319 and an anti-CD47 VH region comprising SEQ ID NO:

320. Claim 24 A bispecific antibody according to claim 23, comprising an anti-CD47 LC region comprising SEQ ID NO: 335 and an anti-CD47 HC region comprising SEQ ID NO:

336. Claim 25 A bispecific antibody according to claim 8, comprising an anti-CD20 VL region comprising SEQ ID NO: 323 and an anti-CD20 VH region comprising SEQ ID NO:

324. Claim 26 A bispecific antibody according to claim 25, comprising an anti-CD20 LC region comprising SEQ ID NO: 331 and an anti-CD20 HC region comprising SEQ ID NO:

332. Claim 27 A bispecific antibody according to claim 10, comprising an anti-CD20 VL region comprising SEQ ID NO: 323 and an anti-CD20 VH region comprising SEQ ID NO:

324. Claim 28 A bispecific antibody according to claim 27, comprising an anti-CD20 LC region comprising SEQ ID NO: 331 and an anti-CD20 HC region comprising SEQ ID NO:

332. Claim 29 A bispecific antibody according to claim 28, comprising an anti-CD47 LC region comprising SEQ ID NO: 333 and an anti-CD47 HC region comprising SEQ ID NO:

334. Claim 30 A bispecific antibody according to claim 28, comprising an anti-CD47 LC region comprising SEQ ID NO: 335 and an anti-CD47 HC region comprising SEQ ID NO:

336. Claim 31 A bispecific antibody according to claim 28, comprising an anti-CD47 LC region comprising SEQ ID NO: 337 and an anti-CD47 HC region comprising SEQ ID NO:

338. Claim 32 A pharmaceutical composition for administration to a patient requiring administration of the pharmaceutical composition for use in a method of treating a tumor, tumor cell, or cancer, comprising: i) the bispecific antibody of claim 1; and ii) at least one pharmaceutically acceptable carrier. Claim 33 In paragraph 32, a pharmaceutical composition in which cancer is a hematological malignant tumor or a B-cell disorder. Claim 34 A pharmaceutical composition according to paragraph 33, wherein the hematological malignancy is a B-cell malignancy. Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete

Images