TROP2 binding protein and its use

Abstract

This specification provides antigen-binding protein constructs (ABPCs) that bind to TROP2 and their use. This specification provides antigen-binding protein constructs (ABPCs) and pharmaceutical compositions comprising an effective amount of ABPC, comprising a first antigen-binding domain (ABD) that can specifically bind to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD at pH about 4.0 to about 6.5 is faster than the dissociation rate at pH about 7.0 to about 8.0, and / or (b) the dissociation constant (KD) of the first ABD at pH about 4.0 to about 6.5 is greater than the KD at pH about 7.0 to about 8.0.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 596,306, filed on 5 November 2023, and U.S. Provisional Patent Application No. 63 / 455,278, filed on 29 March 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application includes a sequence list submitted electronically as an XML file named "45395-0063WO_SL_ST26". The XML file, created on November 4, 2023, is 683,397 bytes in size, and the material contained therein is incorporated herein by reference in its entirety.

[0003] This disclosure relates to the field of antigen-binding molecules. [Overview of the project] [Means for solving the problem]

[0004] The present invention is based on the concept that it is possible to generate antigen-binding protein (ABP) constructs that exhibit enhanced efficacy in toxin release in target mammalian cells, increased endolysosome delivery, and increased cell killing.

[0005] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signaling molecule 2 (TACSTD2), is a cell target of increasing interest in the field of oncology. TROP2 is overexpressed in many large cancers (e.g., NSCLC, breast), and the anti-TROP2 ADC, sacituzumab govitecan (Trodelvy®), is approved for metastatic TNBC. Another anti-TROP2 ADC, datopotamab deruxtecan, has shown promising efficacy in phase 3 trials in HR+ / HER2- cohorts and TNBC cohorts. In particular, Trodelvy has shown lower efficacy in patients with low TROP2 expression compared to patients with high TROP2 expression, indicating an unmet need for patients with tumors that have low TROP2 expression. Furthermore, TROP2 has significant expression in normal tissues, leading to target-mediated clearance of anti-TROP2 ADCs and posing challenges to selectively increasing delivery to tumors rather than normal cells.

[0006] Because manipulating the pH of TROP2 antibodies can potentially reduce the uptake of the corresponding ADC in normal tissue, the applicant has attempted to develop the anti-TROP2 binding proteins disclosed herein. These anti-TROP2 pH-ADCs retain the potential to reduce "on-target" toxicity and broaden the therapeutic concentration range compared to existing non-pH-dependent anti-TROP2 ADCs. The pH-ADCs of this disclosure can also improve efficacy by increasing tumor uptake and improving the PK profile, which may be particularly beneficial in patients with tumors characterized by low TROP2 expression.

[0007] As disclosed herein, the Applicant designed, produced, and evaluated on a large scale approximately 700 antigen-binding protein construct (ABPC) candidates to create a score for ABPCs that exhibit superior pH-dependence, internalization, and cytotoxicity (as ADCs) compared to the corresponding unenhanced control ABPC (or control ADC). Of these initial candidates, nearly 10% showed more than 1.4-fold greater intracellular translocation than their respective control ABPCs, and many of these superior internalizers showed relatively superior cytotoxicity against TROP2-positive cells. Taken together, the results disclosed herein demonstrate that cell TROP2 can be effectively targeted using pH-engineered anti-TROP2 antibodies (and conjugates produced therefrom) that show strong binding at physiological pH but not at the acidic pH found in the endosomal / lysosomal compartment. Ongoing and future studies will evaluate the ability of these enhanced anti-TROP2 ABPCs, particularly with regard to their ability to deliver any desired cargo into TROP2-positive cells both in vitro and in vivo.

[0008] Thus, provided herein are antigen-binding protein constructs (ABPCs) and pharmaceutical compositions comprising an effective amount of an ABPC, the ABPC comprising a first antigen-binding domain (ABD) capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD at a pH of about 4.0 to about 6.5 is faster than the dissociation rate at a pH of about 7.0 to about 8.0, and / or (b) the dissociation constant (KD) of the first ABD at a pH of about 4.0 to about 6.5 is greater than the KD at a pH of about 7.0 to about 8.0.

[0009] In some embodiments, the first ABD comprises the heavy chain variable domain (HCVD) of sacituzumab, datopotamab, hu7E6SVG, or A1,2X4, and each HCVD has at least one (1) histidine substitution (or alanine if histidine was already present).

[0010] As used herein, where a histidine substitution or an alanine substitution is recited, it may be present in one or more of the heavy chain CDRs and / or light chain CDRs, each of which is determined by the Kabat method, IMGT, or the additive "Kabat-IMGT" approach described in Example 1 (i.e., defining a given CDR by including amino acid residues indicated by either or both of the Kabat and IMGT CDR definitions).

[0011] In some embodiments, the first ABD comprises the light chain variable domain (LCVD) of trastuzumab, datopotamab, hu7E6SVG, or A1,2X4's LCVD, and each LCVD has at least one (1) histidine substitution (or alanine if histidine was already present).

[0012] In some embodiments, the first ABD comprises one of (a)-(d): (a) the HCVD of trastuzumab and / or the LCVD of trastuzumab, (b) the HCVD of datopotamab and / or the LCVD of datopotamab, (c) the HCVD of hu7E6SVG and / or the LCVD of hu7E6SVG, and (d) the HCVD of A1,2X4 and / or the LCVD of A1,2X4, and each HCVD or LCVD has one or more amino acids substituted with histidine (or alanine if histidine was already present).

[0013] In some embodiments, the HCVD comprises one of (a)-(d): (a) the HCVD of trastuzumab comprising SEQ ID NO: 4, (b) the HCVD of datopotamab comprising SEQ ID NO: 80, (c) the HCVD of hu7E6SVG comprising SEQ ID NO: 156, and (d) the HCVD of A1,2X4 comprising SEQ ID NO: 234, and each HCVD has one or more amino acids substituted with histidine (or alanine if histidine was already present).

[0014] In some embodiments, the LCVD includes one of the following: (a) to (d): (a) LCVD of sacituzumab containing SEQ ID NO: 5, (b) LCVD of datopotamab containing SEQ ID NO: 81, (c) LCVD of hu7E6SVG containing SEQ ID NO: 157, and (d) LCVD of A1,2X4 containing SEQ ID NO: 235, each LCVD having one or more amino acids substituted with histidine (or alanine in which histidine was already present).

[0015] In some embodiments, the first TROP2 binding domain is (a)~(d): (a) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine; (b) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine. (c) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 158, 159, and 160, and (d) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 236, 237, and 238. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0016] In some embodiments, the first ABD is (a) to (d): (a) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine; (b) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine. (c) LCVDs comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 161, 162, and 163, and (d) LCVDs comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 239, 240, and 241, respectively. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0017] In some embodiments, the first ABD is (a)~(d): (a) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine, (b) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine, (c) SEQ ID NOs. 15 HCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids in total are collectively substituted with histidine, and / or LCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids in total are collectively substituted with histidine, and (d) SEQ ID NOs. The present invention includes one of the following: an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids in total are substituted with histidine; and / or an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids in total are substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0018] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: 80; (c) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, comprising histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156, and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, comprising histidine at one or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0019] In some embodiments, the first ABD is (a)~(d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an LCVD that is at least 90% homologous to SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81; (c) an LCVD that is 95% or 98% homologous to SEQ ID NO: 157, comprising one of the following: (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157; and (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted at the location where histidine was already present.

[0020] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at two or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, containing histidine at two or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: 80. (c) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, comprising histidine at two or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156, and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, comprising histidine at two or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0021] In some embodiments, the first ABD is (a)~(d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at two or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an LCVD that is at least 90%, 95%, homologous to SEQ ID NO: 81, containing histidine at two or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81. (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising histidine at two or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising histidine at two or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0022] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4, and / or selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5 (b) LCVDs that are at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions, (b) HCVDs that are at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: 80, and / or SEQ ID NO: 81, 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and (c) LCVDs containing histidine at one or more positions selected from 95 and at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, (c) HCVDs containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156 and at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, and / or 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93 in SEQ ID NO: 157, (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising histidine at one or more positions selected from 96, 97, 99, 100, and 101; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, comprising histidine at one or more positions in SEQ ID NO: 234; and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted at the location where histidine was already present.

[0023] In some embodiments, the first ABD includes (a) to (d): (a) an HCVD of one of the sequences shown in SEQ ID NOs. 4, 12 to 52, or 313 to 350; (b) an HCVD of one of the sequences shown in SEQ ID NOs. 80, 88 to 128, 397 to 406, or 397 to 406; (c) an HCVD of one of the sequences shown in SEQ ID NOs. 156, 164 to 202, or 427 to 457; and (d) an HCVD of one of the sequences shown in SEQ ID NOs. 234 or 242 to 280. Alternatively, the first ABD includes (a) to (d): (a) an LCVD of one of the sequences shown in SEQ ID NOs. 5, 53 to 79, or one of the sequences shown in SEQ ID NOs. 351 to 396; (b) an LCVD of one of the sequences shown in SEQ ID NOs. 81, 129 to 155, or one of the sequences shown in SEQ ID NOs. 407 to 426; (c) one of the sequences shown in SEQ ID NOs. 157, 203 to 233, or one of the sequences shown in SEQ ID NOs. 458 to 492; and (d) one of the LCVDs of one of the sequences shown in SEQ ID NOs. 235, or one of the sequences shown in SEQ ID NOs. 281 to 312. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0024] In some embodiments, the first ABD is (a)~(d): (a) an HCVD of one of the sequences shown in SEQ ID NOs. 4, 12~52, or 313~350, and / or an LCVD of one of the sequences shown in SEQ ID NOs. 5, 53~79, or 351~396 (where the first ABD is (i) the HCVD of SEQ ID NOs. 4 and the LCVD of SEQ ID NOs. 5, (ii) the HCVD of SEQ ID NOs. 4 and 57, 58, 59, 61, 62, 64, 65, 66, 67, 71, 74, 75, 76, 77, 7 LCVDs other than one of 8, 79, 351, 352, 353, 354, 355, 359, 360, 361, 362, 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 388, 389, 392, 393, 394, 395, and 396, or (iii) LCVD of SEQ ID NO: 5 and SEQ ID NOs: 15, 18, 19, 22, 24, 25, 32, 33, 34, 35, 36, 37, 38, 41, 43, 46, 47, 50, 318, 320, 322, 323, 324, 32 (b) HCVDs that do not include one of the HCVDs that is not one of the following: 5, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, and 348), (b) HCVDs that are one of the sequences shown in sequence numbers 80, 88-128, or 397-406, and / or LCVDs that are one of the sequences shown in sequence numbers 81, 129-155, or 407-426 (where the first ABD is (i) sequence (ii) HCVD of number 80 and LCVD of sequence number 81, (ii) HCVD of sequence number 80 and LCVD other than one of sequence numbers 131, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 147, 151, 152, 153, 411, 412, 417, 418, and 423, or (iii) LCVD of sequence number 81 and HCVD other than one of sequence numbers 91, 101, 103, 112, 113, 116, 119, 122, 123, 125, and 126),(c) One HCVD of the sequences shown in SEQ ID NO: 156 or SEQ ID NOs: 164-202, and / or One LCVD of the sequences shown in SEQ ID NOs: 157, SEQ ID NOs: 203-233, or SEQ ID NOs: 458-492 (where the first ABD is (i) the HCVD of SEQ ID NO: 156 and the LCVD of SEQ ID NO: 157, (ii) an LCVD that is not the HCVD of SEQ ID NO: 156 and one of the sequences shown in SEQ ID NOs: 204, 211, 213, 215, 218, 220, 221, 222, 223, 224, 225, 228, 229, 231, 232, 233, 459, 460, 462, 464, 465, 466, 467, 468, 469, 470, 473, 474, 485, 488, 489, and 491, or (iii) As shown in LCVD of SEQ ID NO: 157, and SEQ ID NOs: 164, 167, 168, 171, 172, 175, 178, 180, 181, 183, 196, 197, 200, 201, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445, 446, 447, 449, 451, 454, 456, and 457. (d) comprising one of the following: (i) an HCVD that is not one of the sequences shown in SEQ ID NO: 234 or SEQ ID NOs: 242-280, and / or one LCVD that is not one of the sequences shown in SEQ ID NO: 235 or SEQ ID NOs: 281-312 (wherein the first ABD does not include the HCVD of SEQ ID NO: 234 and the LCVD of SEQ ID NO: 235). In each of the embodiments described above, alanine may be substituted in place of histidine.

[0025] In some embodiments, ABPC is degraded in target mammalian cells after internal translocation of ABPC by the target mammalian cells. In some embodiments, ABPC comprises a conjugated toxin, radioisotope, drug, or small molecule.

[0026] In some embodiments, the composition provides increased toxin release in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least 20% compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least 50% compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least twofold compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least fivefold compared to a composition containing the same amount of control ABPC.

[0027] In some embodiments, the composition provides an increased killing of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least 20% or 50% in killing of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least 2 to 5 times in killing of target mammalian cells compared to a composition containing the same amount of control ABPC.

[0028] In some embodiments, the composition provides an increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least 20% to 50% in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least two or five times in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0029] In some embodiments, the composition results in a less significant decrease in the level of TROP2 presented on the surface of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition results in no detectable decrease in the level of TROP2 presented on the surface of target mammalian cells.

[0030] Also provided herein is a pharmaceutical composition comprising an effective amount of antigen-binding protein construct (ABPC), the first ABD capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug, or small molecule, wherein (a) the dissociation rate of the first ABD at pH about 4.0 to about 6.5 is faster than the dissociation rate at pH about 7.0 to about 8.0, or the dissociation constant (KD) of the first ABD at pH about 4.0 to about 6.5 is greater than the KD at pH about 7.0 to about 8.0, and (b) the composition provides one or more of the following compared to a composition containing the same amount of control ABPC: increased toxin release in target mammalian cells, increased killing of target mammalian cells compared to a composition containing the same amount of control ABPC, and increased endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0031] In some embodiments, the first ABD comprises an HCVD which is one of the following: (a) HCVD of sacituzumab, (b) HCVD of datopotamab, (c) HCVD of hu7E6SVG, and (d) HCVD of A1,2X4, each HCVD having one or more histidine substitutions. In some embodiments, the first ABD comprises an LCVD which is one of the following: (a) LCVD of sacituzumab, (b) LCVD of datopotamab, (c) LCVD of hu7E6SVG, and (d) LCVD of A1,2X4, each LCVD having one or more histidine substitutions. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0032] In some embodiments, the first ABD comprises one of the following: (a) to (d): (a) HCVD and / or LCVD of sacituzumab, (b) HCVD and / or LCVD of datopotamab, (c) HCVD and / or LCVD of hu7E6SVG, and (d) HCVD and / or LCVD of A1,2X4, where each HCVD and / or LCVD has one or more histidine substitutions. In each of the embodiments described above, alanine may be substituted in place of histidine that was already present.

[0033] In some embodiments, the HCVD includes one of the following: (a) to (d): (a) the HCVD of sacituzumab including SEQ ID NO: 4, (b) the HCVD of datopotamab including SEQ ID NO: 80, (c) the HCVD of hu7E6SVG including SEQ ID NO: 156, and (d) the HCVD of A1,2X4 including SEQ ID NO: 234. In some embodiments, the LCVD includes one of the following: (a) to (d): (a) the LCVD of sacituzumab including SEQ ID NO: 5, (b) the LCVD of datopotamab including SEQ ID NO: 81, (c) the LCVD of hu7E6SVG including SEQ ID NO: 157, and (d) the LCVD of A1,2X4 including SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0034] In some embodiments, the first ABD is (a)~(d): (a) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 6, 7, and 8; (b) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 82, 83, and 84; (c) sequence number The present invention includes an HCVD comprising (d) an HCVD comprising one of the following: (d) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, in which one or more amino acids are collectively substituted with histidine; and (d) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, in which one or more amino acids are collectively substituted with histidine; in each of the embodiments described above, alanine may be substituted in place of histidine.

[0035] In some embodiments, the first ABD is (a) to (d): (a) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 9, 10, and 11; (b) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 85, 86, and 87; (c) (d) LCVDs comprising (d) LCVDs comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids are collectively substituted with histidine, and (d) LCVDs comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids are collectively substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0036] In some embodiments, the first ABD is (a)~(d): (a) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine, (b) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine, (c) SEQ ID NOs. 15 HCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids in total are collectively substituted with histidine, and / or LCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids in total are collectively substituted with histidine, and (d) SEQ ID NOs. The present invention includes one of the following: an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids in total are substituted with histidine; and / or an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids in total are substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0037] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at one or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0038] In some embodiments, the first ABD is (a) to (d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising a histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and comprising one LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising a histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0039] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at two or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an SEQ ID NO: 80, containing histidine at two or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at two or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at two or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0040] In some embodiments, the first ABD is (a) to (d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at two or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an SEQ ID NO: 81, containing histidine at two or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising a histidine at two or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and comprising one LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising a histidine at two or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0041] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4, and / or selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5 (b) LCVDs that are at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions; (b) HCVDs that are at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: 80, and / or 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 9 in SEQ ID NO: 81 (c) LCVDs containing histidine at one or more positions selected from 5, which are at least 90%, 95%, or 98% homologous to SEQ ID NO: 81; (c) HCVDs containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156, which are at least 90%, 95%, or 98% homologous to SEQ ID NO: 156; and / or SEQ ID NO: 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 9 in SEQ ID NO: 157 (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, containing histidine at one or more positions selected from 6, 97, 99, 100, and 101; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at one or more positions in SEQ ID NO: 234; and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, containing histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted at the location where histidine was already present.

[0042] In some embodiments, the first ABD includes an HCVD which is one of the following: (a) to (d): (a) an HCVD which is one of the sequences shown in SEQ ID NOs. 4, 12 to 52, or 313 to 350; (b) an HCVD which is one of the sequences shown in SEQ ID NOs. 80, 88 to 128, 397 to 406, or 397 to 406; (c) an HCVD which is one of the sequences shown in SEQ ID NOs. 156, 164 to 202, or 427 to 457; and (d) an HCVD which is one of the sequences shown in SEQ ID NOs. 234, or 242 to 280. In some embodiments, the first ABD includes one LCVD from among (a) to (d): (a) one LCVD of one of the sequences shown in SEQ ID NOs. 5, 53 to 79, or 351 to 396; (b) one LCVD of one of the sequences shown in SEQ ID NOs. 81, 129 to 155, or 407 to 426; (c) one LCVD of one of the sequences shown in SEQ ID NOs. 157, 203 to 233, or 458 to 492; and (d) one LCVD of one of the sequences shown in SEQ ID NOs. 235, or 281 to 312.

[0043] In some embodiments, the first ABD is (a)~(d): (a) Sequence ID 4, Sequence ID HCVD of one of the sequences shown in 12-52, or one of the sequences shown in SEQ ID NOs. 313-350, and / or LCVD of one of the sequences shown in SEQ ID NOs. 5, 53-79, or 351-396 (where the first ABD is (i) the HCVD of SEQ ID NOs. 4 and the LCVD of SEQ ID NOs. 5, (ii) the HCVD of SEQ ID NOs. 4, and SEQ ID NOs. 57, 58, 59, 61, 62, 64, 65, 66, 67, 71, 74, 75, 76, 77, 78, 79, 351, 352, 353, 354, 355, 359, 360, 361, 362, 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 3 (iii) an LCVD that is not one of 88, 389, 392, 393, 394, 395, and 396, or (iii) an HCVD that is not one of 15, 18, 19, 22, 24, 25, 32, 33, 34, 35, 36, 37, 38, 41, 43, 46, 47, 50, 318, 320, 322, 323, 324, 325, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, and 348, and does not include the LCVD of SEQ ID NO. 5), (b) an HCVD that is not one of the sequences shown in SEQ ID NO. 80, one of the sequences shown in SEQ ID NOs. 88-128, one of the sequences shown in SEQ ID NOs. 397-406, or one of the sequences shown in SEQ ID NOs. 397-406, and / or an HCVD that is not one of the sequences shown in SEQ ID NOs. 81, SEQ ID NOs. LCVD of one of the sequences shown in 129-155, or one of the sequences shown in sequence numbers 407-426 (where the first ABD is (i) the HCVD of sequence number 80 and the LCVD of sequence number 81, (ii) the HCVD of sequence number 80, and sequence numbers 131, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 147, 151, 152, 153, 411, 412, 417, 418,(iii) an LCVD that is not one of the sequences in sequence numbers 91, 101, 103, 112, 113, 116, 119, 122, 123, 125, and 126, and not the LCVD of sequence number 81), (c) an HCVD that is not one of the sequences shown in sequence number 156, sequence numbers 164-202, or one of the sequences shown in sequence numbers 427-457, and / or sequence number 157, sequence numbers 203-233 One of the sequences shown in or one of the sequences shown in sequence numbers 458-492 is an LCVD (where the first ABD is (i) the HCVD of sequence number 156 and the LCVD of sequence number 157, (ii) the HCVD of sequence number 156, and sequence numbers 204, 211, 213, 215, 218, 220, 221, 222, 223, 224, 225, 228, 229, 231, 232, 233, 459, 460, 462, 464, 465, 466, 467, 468 LCVD that is not one of the sequences shown in 469, 470, 473, 474, 485, 488, 489, and 491, or (iii) SEQ ID NOs: 164, 167, 168, 171, 172, 175, 178, 180, 181, 183, 196, 197, 200, 201, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445, 446, 447, 449, 451, 454, 4 (d) containing one of the following: (d) an HCVD that is not one of the sequences shown in sequence numbers 56, and 457, and not the LCVD of sequence number 157; and (d) one of the HCVDs shown in sequence number 234, or sequences 242-280, and / or one of the LCVDs shown in sequence number 235, or sequences 281-312 (where the first ABD does not contain the HCVD of sequence number 234, or the LCVD of sequence number 235).

[0044] In some embodiments, the composition provides increased toxin release in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least 20% or 50% compared to a composition containing the same amount of control ABPC. In some embodiments, the composition increases toxin release in target mammalian cells by at least twofold or fivefold compared to a composition containing the same amount of control ABPC.

[0045] In some embodiments, the composition provides an increased killing of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least 20% or 50% in killing of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides an increase of at least 2x or 5x in killing of target mammalian cells compared to a composition containing the same amount of control ABPC.

[0046] In some embodiments, the composition provides an increase in endolysosome delivery, including an increase of at least 20% or 50% in endolysosome delivery compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides at least a twofold or fivefold increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0047] In some embodiments, the composition results in a smaller reduction in the level of TROP2 presented on the surface of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition results in no detectable reduction in the level of TROP2 presented on the surface of target mammalian cells. In some embodiments, the target mammalian cells are cancer cells.

[0048] In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 10% faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 3 times faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 10 times faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the K of ABD at pH approximately 4.0 to approximately 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least 10% greater than that. In some embodiments, the K of ABD at pH about 4.0 to about 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least three times larger than that. In some embodiments, the K of ABD at pH about 4.0 to about 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least 10 times larger than that.

[0049] In some embodiments, ABPC is cytotoxic or inhibits cell proliferation to target mammalian cells.

[0050] In some embodiments, ABPC is cross-reactive with non-human primate TROP2 and human TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, and one or both of rat TROP2 and mouse TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, rat TROP2, and mouse TROP2. In some embodiments, ABD binds to TROP2 epitopes present on the surface of cells from Old World monkeys.

[0051] In some embodiments, the ABPC comprises a single polypeptide. In some embodiments, the ABD is selected from a VH domain, a VHH domain, a VNAR domain, and scFv. In some embodiments, the ABPC is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scdiabody, scdiabody-CH3, scFv-CH-CL-scFv, HSAbody, scdiabody-HSA, or tandem-scFv.

[0052] In some embodiments, ABPC comprises two or more polypeptides. In some embodiments, ABPC may include antibody, VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF(two-in-one), DAF(four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, VHH-Fc, Tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, Dock and Lock The following are selected from the group of (lock), ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adonectin, DARPin, Fibronectin, DEP conjugate, PROTAB, and PROTAC.

[0053] In some embodiments, ABPC or ABD includes a proteolytically targeted antibody (PROTAB) (e.g., as described in Marei et al., “Antibody targeting of E3 ubiquitin ligases for receptor degradation.” Nature, 6 October 2022). In some embodiments, PROTAB or PROTAC may anchor cell surface E3 ubiquitin ligases to transmembrane proteins, resulting in targeted degradation both in vitro and in vivo.

[0054] In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a cleavable linker. In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a non-cleavable linker.

[0055] In some embodiments, the in vivo half-life of ABPC increases compared to the in vivo half-life of control ABPC. In some embodiments, the in vivo half-life of ABPC increases by approximately 5% to approximately 95% compared to the in vivo half-life of control ABPC. In some embodiments, the in vivo half-life of ABPC increases by approximately 10% to approximately 95% compared to the in vivo half-life of control ABPC. In some embodiments, the in vivo half-life of ABPC increases by approximately 30% to approximately 95% compared to the in vivo half-life of control ABPC. In some embodiments, the in vivo half-life of ABPC increases by approximately 50% to approximately 95% compared to the in vivo half-life of control ABPC. In some embodiments, the in vivo half-life of ABPC increases by approximately 70% to approximately 95% compared to the in vivo half-life of control ABPC. In other embodiments, the in vivo half-life can be shortened.

[0056] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, (a) the control ABPC comprises a first ABD; (b) the dissociation rate of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 3-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K D ) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 3-fold greater than K D at a pH of about 7.0 to about 8.0.

[0057] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, (a) the control ABPC comprises a first ABD; (b) the dissociation rate of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 2-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K D ) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 2-fold greater than K D at a pH of about 7.0 to about 8.0.

[0058] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, (a) the control ABPC comprises a first ABD; (b) the dissociation rate of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 1-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K D ) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than 1-fold greater than K D at a pH of about 7.0 to about 8.0.

[0059] In some embodiments, the control ABPC is selected from one of (a) trastuzumab, (b) datopotamab, (b) hu7E6SVG, and (d) A1,2X4.

[0060] In some embodiments, the ABPC includes a second ABD.

[0061] Furthermore, provided herein are kits comprising at least one dose of any of the pharmaceutical compositions described herein.

[0062] Furthermore, this specification provides for (a) a first ABD whose dissociation rate at pH approximately 4.0 to approximately 6.5 is faster than the dissociation rate at pH approximately 7.0 to approximately 8.0, or (b) a dissociation constant (K) of the first ABD at pH approximately 4.0 to approximately 6.5. D ) is K at a pH of approximately 7.0 to 8.0. D This is a first antigen-binding protein construct (ABPC) containing an ABD that is larger than the target mammalian cell and can specifically bind to TROP2 or an epitope of TROP2 presented on the surface of the target mammalian cell.

[0063] In some embodiments, the first ABD includes one of the following: (a) to (d): (a) an HCVD of sacituzumab having one or more amino acids substituted with histidine, (b) an HCVD of datopotamab having one or more amino acids substituted with histidine, (c) an HCVD of hu7E6SVG having one or more amino acids substituted with histidine, and (d) an HCVD of A1,2X4 having one or more amino acids substituted with histidine. In some embodiments, the first ABD includes one of the following: (a) to (d): (a) an LCVD of sacituzumab having one or more amino acids substituted with histidine, (b) an LCVD of datopotamab having one or more amino acids substituted with histidine, (c) an LCVD of hu7E6SVG having one or more amino acids substituted with histidine, and (d) an LCVD of A1,2X4 having one or more amino acids substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0064] In some embodiments, the first ABD includes one of the following: (a) to (d): (a) HCVD of sacituzumab and / or LCVD of sacituzumab (either or both having one or more amino acids substituted with histidine), (b) HCVD of datopotamab and / or LCVD of datopotamab (either or both having one or more amino acids substituted with histidine), (c) HCVD of hu7E6SVG and / or LCVD of hu7E6SVG (either or both having one or more amino acids substituted with histidine), and (d) HCVD of A1,2X4 and / or LCVD of A1,2X4 (either or both having one or more amino acids substituted with histidine). In some embodiments, the HCVD includes one of the following: (a) to (d): (a) the HCVD of sacituzumab including SEQ ID NO: 4, (b) the HCVD of datopotamab including SEQ ID NO: 80, (c) the HCVD of hu7E6SVG including SEQ ID NO: 156, and (d) the HCVD of A1,2X4 including SEQ ID NO: 234. In some embodiments, the LCVD includes one of the following: (a) to (d): (a) the LCVD of sacituzumab including SEQ ID NO: 5, (b) the LCVD of datopotamab including SEQ ID NO: 81, (c) the LCVD of hu7E6SVG including SEQ ID NO: 157, and (d) the LCVD of A1,2X4 including SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0065] In some embodiments, the first ABD is (a)~(d): (a) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 6, 7, and 8; (b) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 82, 83, and 84; (c) sequence number (d) an HCVD comprising one of the following: (a) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, in which one or more amino acids are collectively substituted with histidine, respectively; and (d) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, in which one or more amino acids are collectively substituted with histidine, respectively. In some embodiments, the first ABD comprises (a) to (d): (a) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, in which one or more amino acids are collectively substituted with histidine, respectively; (b) (c) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine; (a) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids are collectively substituted with histidine; and (d) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids are collectively substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0066] In some embodiments, the first ABD is (a)~(d): (a) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine, (b) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine, (c) SEQ ID NOs. 15 HCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids in total are collectively substituted with histidine, and / or LCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids in total are collectively substituted with histidine, and (d) SEQ ID NOs. The present invention includes one of the following: an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids in total are substituted with histidine; and / or an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids in total are substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0067] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at one or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0068] In some embodiments, the first ABD is (a)~(d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, containing histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, containing histidine at one or more positions in SEQ ID NO: 235.

[0069] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at two or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an SEQ ID NO: 80, containing histidine at two or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at two or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at two or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0070] In some embodiments, the first ABD is (a)~(d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at two or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, containing histidine at two or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 The LCVD is one of the following: (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, containing histidine at two or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157; and (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, containing histidine at two or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0071] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4, and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5, (b) SEQ ID NO: SEQ ID NO: 80 contains histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108. (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80, and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81, (c) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156, (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, comprising histidine at one or more positions in SEQ ID NO: 234; and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted at the location where histidine was already present.

[0072] In some embodiments, the first ABD includes an HCVD which is one of the following: (a)~(d): (a) one of the sequences shown in SEQ ID NO: 4, SEQ ID NOs: 12~52, or one of the sequences shown in SEQ ID NOs: 313~350; (b) one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88~128, one of the sequences shown in SEQ ID NOs: 397~406, or one of the sequences shown in SEQ ID NOs: 397~406; (c) one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164~202, or one of the sequences shown in SEQ ID NOs: 427~457; and (d) one of the sequences shown in SEQ ID NO: 234, or one of the sequences shown in SEQ ID NOs: 242~280, and / or the first ABD includes an HCVD which is one of the following: (a)~(d): (a) SEQ ID NO: 5, SEQ ID NO: (b) LCVD of one of the sequences shown in 53-79 or one of the sequences shown in SEQ ID NOs: 351-396, (b) LCVD of one of the sequences shown in SEQ ID NOs: 81, 129-155 or one of the sequences shown in SEQ ID NOs: 407-426, (c) LCVD of one of the sequences shown in SEQ ID NOs: 157, 203-233 or one of the sequences shown in SEQ ID NOs: 458-492, and (d) LCVD of one of the sequences shown in SEQ ID NOs: 235 or one of the sequences shown in SEQ ID NOs: 281-312. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0073] In some embodiments, the first ABD is (a)~(d): (a) an HCVD of one of the sequences shown in SEQ ID NOs. 4, 12~52, or 313~350, and / or an LCVD of one of the sequences shown in SEQ ID NOs. 5, 53~79, or 351~396 (where the first ABD is (i) the HCVD of SEQ ID NOs. 4 and the LCVD of SEQ ID NOs. 5, (ii) the HCVD of SEQ ID NOs. 4, and SEQ ID NOs. 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 71, 74, 75, 76, 77, 78, LCVD other than one of 79, 351, 352, 353, 354, 355, 359, 360, 361, 362, 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 388, 389, 392, 393, 394, 395, and 396, or (iii) Sequence IDs 15, 18, 19, 22, 24, 25, 32, (b) HCVDs that are not one of 33, 34, 35, 36, 37, 38, 41, 43, 46, 47, 50, 318, 320, 322, 323, 324, 325, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, and 348, and LCVD of sequence number 5), (b) HCVDs of one of the sequences shown in sequence number 80, sequence numbers 88-128, sequence numbers 397-406, or sequence numbers 397-406, and / or LCVDs of one of the sequences shown in sequence number 81, sequence numbers 129-155, or sequence numbers 407-426 (where the first ABD is (i) HCVD of sequence number 80 and LCVD of sequence number 81, (ii) sequence number(iii) HCVDs of 80, and LCVDs other than one of SEQ ID NOs: 131, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 147, 151, 152, 153, 411, 412, 417, 418, and 423, or (iii) HCVDs other than one of SEQ ID NOs: 91, 101, 103, 112, 113, 116, 119, 122, 123, 125, and 126, and LCVD of SEQ ID NO: 81, (c) those shown in SEQ ID NOs: 156, 164-202 HCVD of one of the sequences, or one of the sequences shown in sequence numbers 427-457, and / or LCVD of one of the sequences shown in sequence number 157, sequence numbers 203-233, or one of the sequences shown in sequence numbers 458-492 (where the first ABD is (i) the HCVD of sequence number 156 and the LCVD of sequence number 157, (ii) the HCVD of sequence number 156, and sequence numbers 204, 211, 213, 215, 218, 220, 221, 222, 223, 224, 225, 228 LCVD that is not one of the sequences shown in 229, 231, 232, 233, 459, 460, 462, 464, 465, 466, 467, 468, 469, 470, 473, 474, 485, 488, 489, and 491, or (iii) LCVD that is not one of the sequences shown in SEQ ID NOs: 164, 167, 168, 171, 172, 175, 178, 180, 181, 183, 196, 197, 200, 201, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 4 (d) including one of the HCVD sequences that are not one of the sequences shown in 45, 446, 447, 449, 451, 454, 456, and 457, and the LCVD of sequence number 157), and (d) including one of the HCVD sequences shown in sequence number 234 or sequence numbers 242-280, and / or one of the LCVD sequences shown in sequence number 235 or sequence numbers 281-312 (where the first ABD does not include the HCVD of sequence number 234 and the LCVD of sequence number 235).

[0074] In some embodiments, ABPC is degraded in target mammalian cells after internal translocation of ABPC by the target mammalian cells. In some embodiments, ABPC comprises a conjugated toxin, radioisotope, drug, or small molecule.

[0075] In some embodiments, compositions containing ABPC provide increased toxin release in target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least 20% or 50% compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least twofold or fivefold compared to a control composition containing the same amount of ABPC.

[0076] In some embodiments, a composition containing ABPC provides increased killing of target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, a composition containing ABPC provides at least a 20% or 50% increase in killing of target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, a composition containing ABPC provides at least a 2-fold or 5-fold increase in killing of target mammalian cells compared to a control composition containing the same amount of ABPC.

[0077] In some embodiments, a composition containing ABPC provides an increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, ABPC provides at least a 20% increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, a composition containing ABPC provides at least a 50% increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, a composition containing ABPC provides at least a twofold increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, a composition containing ABPC provides at least a fivefold increase in endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0078] In some embodiments, compositions containing ABPC result in a smaller reduction in the level of TROP2 presented on the surface of target mammalian cells compared to compositions containing the same amount of control ABPC. In some embodiments, compositions containing ABPC do not result in a detectable reduction in the level of TROP2 presented on the surface of target mammalian cells.

[0079] Furthermore, in this specification, an antigen-binding protein construct (ABPC) comprising a first ABD that can specifically bind to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug, or small molecule, wherein (a) the dissociation rate of the first ABD at pH approximately 4.0 to approximately 6.5 is faster than the dissociation rate at pH approximately 7.0 to approximately 8.0, or the dissociation constant (K) of the first ABD at pH approximately 4.0 to approximately 6.5. D ) is K at pH approximately 7.0 to 8.0 DA larger antigen-binding protein construct (ABPC) is provided, wherein (b) the composition provides one or more of the following: increased toxin release in target mammalian cells compared to a composition containing the same amount of control ABPC; increased killing of target mammalian cells compared to a composition containing the same amount of control ABPC; and increased endolysosome delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0080] In some embodiments, the first ABD comprises one of the following: (a) HCVD of sacituzumab, (b) HCVD of datopotamab, (c) HCVD of hu7E6SVG, and (d) HCVD of A1,2X4, each or any of which has one or more amino acids substituted with histidine. In some embodiments, the first ABD comprises one of the following: (a) LCVD of sacituzumab, (b) LCVD of datopotamab, (c) LCVD of hu7E6SVG, and (d) LCVD of A1,2X4, each or any of which has one or more amino acids substituted with histidine. In some embodiments, the first ABD comprises one of the following: (a) to (d): (a) HCVD and LCVD of sacituzumab, (b) HCVD and LCVD of datopotamab, (c) HCVD and LCVD of hu7E6SVG, and (d) HCVD and LCVD of A1,2X4, where each or any of the domains has one or more amino acids substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0081] In some embodiments, the HCVD includes one of the following: (a) to (d): (a) the HCVD of sacituzumab including SEQ ID NO: 4, (b) the HCVD of datopotamab including SEQ ID NO: 80, (c) the HCVD of hu7E6SVG including SEQ ID NO: 156, and (d) the HCVD of A1,2X4 including SEQ ID NO: 234. In some embodiments, the LCVD includes one of the following: (a) to (d): (a) the LCVD of sacituzumab including SEQ ID NO: 5, (b) the LCVD of datopotamab including SEQ ID NO: 81, (c) the LCVD of hu7E6SVG including SEQ ID NO: 157, and (d) the LCVD of A1,2X4 including SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0082] In some embodiments, the first ABD is (a)~(d): (a) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 6, 7, and 8; (b) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 82, 83, and 84; (c) sequence number (d) an HCVD comprising one of the HCVDs comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids are collectively substituted with histidine, and (d) an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids are collectively substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0083] In some embodiments, the first ABD is (a) to (d): (a) LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine in SEQ ID NOs. 9, 10, and 11; (b) (c) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine; (a) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids are collectively substituted with histidine; and (d) an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids are collectively substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0084] In some embodiments, the first ABD is (a)~(d): (a) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, in which one or more amino acids are collectively substituted with histidine, (b) an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, in which one or more amino acids are collectively substituted with histidine, and / or an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, in which one or more amino acids are collectively substituted with histidine, (c) SEQ ID NOs. 15 HCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, in which one or more amino acids in total are collectively substituted with histidine, and / or LCVDs containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, in which one or more amino acids in total are collectively substituted with histidine, and (d) SEQ ID NOs. The present invention includes one of the following: an HCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, in which one or more amino acids in total are substituted with histidine; and / or an LCVD comprising CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, in which one or more amino acids in total are substituted with histidine. In each of the embodiments described above, alanine may be substituted in place of histidine.

[0085] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4; (b) an SEQ ID NO: 80, containing histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 in SEQ ID NO: The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at one or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0086] In some embodiments, the first ABD is (a) to (d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising a histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and comprising one LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising a histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0087] In some embodiments, the first ABD is (a) to (d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at two or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 8 The HCVD is one of the following: (c) an HCVD that is at least 90%, 95%, or 98% homologous to 0; (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at two or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, containing histidine at two or more positions in SEQ ID NO: 234. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0088] In some embodiments, the first ABD is (a) to (d): (a) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at two or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5; (b) an SEQ ID NO: 81, containing histidine at two or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81 (c) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising a histidine at two or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157, and comprising one LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising a histidine at two or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted in the place where histidine was already present.

[0089] In some embodiments, the first ABD is (a)~(d): (a) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, containing histidine at one or more positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4, and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, containing histidine at one or more positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5, (b) SEQ ID NO: SEQ ID NO: 80 contains histidine at one or more positions selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108. (c) an HCVD that is at least 90%, 95%, or 98% homologous to 80, and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, containing histidine at one or more positions selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95 in SEQ ID NO: 81, (c) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, containing histidine at one or more positions selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 in SEQ ID NO: 156, (d) an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, comprising histidine at one or more positions selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101 in SEQ ID NO: 157; and (d) an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, comprising histidine at one or more positions in SEQ ID NO: 234; and / or an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, comprising histidine at one or more positions in SEQ ID NO: 235. In each of the embodiments described above, alanine may be substituted at the location where histidine was already present.

[0090] In some embodiments, the first ABD includes one of the following: (a) to (d): (a) an HCVD of one of the sequences shown in SEQ ID NOs. 4, 12 to 52, or 313 to 350; (b) an HCVD of one of the sequences shown in SEQ ID NOs. 80, 88 to 128, 397 to 406, or 397 to 406; (c) an HCVD of one of the sequences shown in SEQ ID NOs. 156, 164 to 202, or 427 to 457; and (d) an HCVD of one of the sequences shown in SEQ ID NOs. 234, or 242 to 280. In some embodiments, the first ABD includes one of the following: (a) to (d): (a) an LCVD of one of the sequences shown in SEQ ID NOs. 5, 53 to 79, or 351 to 396; (b) an LCVD of one of the sequences shown in SEQ ID NOs. 81, 129 to 155, or 407 to 426; (c) an LCVD of one of the sequences shown in SEQ ID NOs. 157, 203 to 233, or 458 to 492; and (d) an LCVD of one of the sequences shown in SEQ ID NOs. 235, or 281 to 312.

[0091] In some embodiments, the first ABD is (a)~(d): (a) Sequence ID 4, Sequence ID HCVD of one of the sequences shown in 12-52, or one of the sequences shown in sequence numbers 313-350, and / or LCVD of one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396 (where the first ABD is (i) the HCVD of sequence number 4 and the LCVD of sequence number 5, (ii) the HCVD of sequence number 4, and sequence numbers 57, 58, 59, 61, 62, 64, 65, 66, 67, 71, 74, 75, 76, 77, 78, 79, 351, 352, 353, 354, 355, 359, 360, 361, 362, 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 38 (iii) an LCVD that is not one of sequence numbers 8, 389, 392, 393, 394, 395, and 396, or (iii) an HCVD that is not one of sequence numbers 15, 18, 19, 22, 24, 25, 32, 33, 34, 35, 36, 37, 38, 41, 43, 46, 47, 50, 318, 320, 322, 323, 324, 325, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, and 348, and does not include the LCVD of sequence number 5), (b) an HCVD that is not one of the sequences shown in sequence number 80, sequence numbers 88-128, one of the sequences shown in sequence numbers 397-406, or one of the sequences shown in sequence numbers 397-406, and / or sequence number 81, sequence number LCVD of one of the sequences shown in 129-155, or one of the sequences shown in sequence numbers 407-426 (where the first ABD is (i) the HCVD of sequence number 80 and the LCVD of sequence number 81, (ii) the HCVD of sequence number 80, and sequence numbers 131, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 147, 151, 152, 153, 411, 412, 417, 418,(iii) an LCVD that is not one of the sequences in sequence numbers 91, 101, 103, 112, 113, 116, 119, 122, 123, 125, and 126, and not the LCVD of sequence number 81), (c) an HCVD that is one of the sequences shown in sequence number 156, sequence numbers 164-202, or one of the sequences shown in sequence numbers 427-457, and / or sequence number 157, sequence number 203 LCVD of one of the sequences shown in ~233, or one of the sequences shown in sequence numbers 458~492 (where the first ABD is (i) the HCVD of sequence number 156 and the LCVD of sequence number 157, (ii) the HCVD of sequence number 156, and sequence numbers 204, 211, 213, 215, 218, 220, 221, 222, 223, 224, 225, 228, 229, 231, 232, 233, 459, 460, 462, 464, 465, 466) LCVD that is not one of the sequences shown in 467, 468, 469, 470, 473, 474, 485, 488, 489, and 491, or (iii) Sequence IDs 164, 167, 168, 171, 172, 175, 178, 180, 181, 183, 196, 197, 200, 201, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445, 446, 447, (d) containing one of the HCVDs that is not one of the sequences shown in 449, 451, 454, 456, and 457, and one of the LCVDs of sequence number 157), and (d) containing one of the HCVDs that is one of the sequences shown in sequence number 234, or sequence numbers 242-280, and / or one of the LCVDs that is one of the sequences shown in sequence number 235, or sequence numbers 281-312 (where the first ABD does not contain the HCVD of sequence number 234).

[0092] In some embodiments, compositions containing ABPC provide increased toxin release in target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least 20% compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least 50% compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least twofold compared to a control composition containing the same amount of ABPC. In some embodiments, compositions containing ABPC increase toxin release in target mammalian cells by at least fivefold compared to a control composition containing the same amount of ABPC.

[0093] In some embodiments, a composition containing ABPC provides increased killing of target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, a composition containing ABPC provides at least a 20% or 50% increase in killing of target mammalian cells compared to a control composition containing the same amount of ABPC. In some embodiments, a composition containing ABPC provides at least a 2-fold or 5-fold increase in killing of target mammalian cells compared to a control composition containing the same amount of ABPC.

[0094] In some embodiments, compositions containing ABPC provide increased endolysosome delivery in target mammalian cells compared to compositions containing the same amount of control ABPC. In some embodiments, compositions containing ABPC provide at least a 20% or 50% increase in endolysosome delivery in target mammalian cells compared to compositions containing the same amount of control ABPC. In some embodiments, compositions containing ABPC provide at least a two-fold or five-fold increase in endolysosome delivery in target mammalian cells compared to compositions containing the same amount of control ABPC.

[0095] In some embodiments, compositions containing ABPC result in a smaller reduction in the level of TROP2 presented on the surface of target mammalian cells compared to compositions containing the same amount of control ABPC. In some embodiments, compositions containing ABPC result in no detectable reduction in the level of TROP2 presented on the surface of target mammalian cells. In some embodiments, the target mammalian cells are cancer cells.

[0096] In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 10% faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 3 times faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the dissociation rate of ABD at pH approximately 4.0 to approximately 6.5 is at least 10 times faster than the dissociation rate of ABD at pH approximately 7.0 to approximately 8.0. In some embodiments, the K of ABD at pH approximately 4.0 to approximately 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least 10% greater than that. In some embodiments, the K of ABD at pH about 4.0 to about 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least three times larger than that. In some embodiments, the K of ABD at pH about 4.0 to about 6.5 D This is the K of ABD at a pH of approximately 7.0 to 8.0. D It is at least 10 times larger than that.

[0097] In some embodiments, ABPC is cytotoxic or inhibits cell proliferation to target mammalian cells.

[0098] In some embodiments, ABPC is cross-reactive with non-human primate TROP2 and human TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, and one or both of rat TROP2 and mouse TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, rat TROP2, and mouse TROP2. In some embodiments, ABD binds to TROP2 epitopes present on the surface of cells from Old World monkeys.

[0099] In some embodiments, the ABPC comprises a single polypeptide. In some embodiments, the ABD is selected from a VH domain, a VHH domain, a VNAR domain, and scFv. In some embodiments, the ABPC is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scdiabody, scdiabody-CH3, scFv-CH-CL-scFv, HSAbody, scdiabody-HSA, or tandem-scFv.

[0100] In some embodiments, ABPC comprises two or more polypeptides. In some embodiments, ABPC may include antibody, VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF(two-in-one), DAF(four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, VHH-Fc, Tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, Dock and Lock The following are selected from the group of (lock), ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adonectin, DARPin, Fibronectin, DEP conjugate, PROTAB, and PROTAC.

[0101] In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a cleavable linker. In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a non-cleavable linker.

[0102] In some embodiments, the half-life of ABPC in vivo is reduced compared to the half-life of control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is reduced by approximately 5% to 95% compared to the half-life of control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is reduced by approximately 10% to 95% compared to the half-life of control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is reduced by approximately 30% to 95% compared to the half-life of control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is reduced by approximately 50% to 95% compared to the half-life of control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is reduced by approximately 70% to 95% compared to the half-life of control ABPC in vivo.

[0103] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of target mammalian cells, wherein (a) the control ABPC contains a primary ABD; (b) the dissociation rate of the primary ABD of the control ABPC at pH approximately 4.0 to approximately 6.5 is no more than 3 times faster than the dissociation rate at pH approximately 7.0 to approximately 8.0; and (c) the dissociation constant (K) of the primary ABD of the control ABPC at pH approximately 4.0 to approximately 6.5. D ) is K at a pH of approximately 7.0 to 8.0. D It is less than three times larger.

[0104] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of target mammalian cells, wherein (a) the control ABPC contains a primary ABD; (b) the dissociation rate of the primary ABD of the control ABPC at pH approximately 4.0 to approximately 6.5 is no more than twice as fast as the dissociation rate at pH approximately 7.0 to 8.0; and (c) the dissociation constant (K) of the primary ABD of the ABPC at pH approximately 4.0 to 6.5. D ) is K at a pH of approximately 7.0 to 8.0. D It is less than twice as large.

[0105] In some embodiments, the control ABPC is capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of target mammalian cells, wherein (a) the control ABPC contains a primary ABD; (b) the dissociation rate of the primary ABD of the control ABPC at pH approximately 4.0 to approximately 6.5 is less than 1 times faster than the dissociation rate at pH approximately 7.0 to approximately 8.0; and (c) the dissociation constant (K) of the primary ABD of the control ABPC at pH approximately 4.0 to approximately 6.5. D ) is K at a pH of approximately 7.0 to 8.0. D It is less than 1 times larger than that.

[0106] In some embodiments, the control ABPC is sacituzumab, datopotamab, hu7E6SVG, or A1,2X4.

[0107] In some embodiments, the ABPC includes a second ABD.

[0108] Furthermore, a kit comprising at least one dose of any ABPC described herein is provided herein.

[0109] Furthermore, this specification provides a method for treating cancer characterized by having a population of cancer cells having TROP2 or an epitope of TROP2 presented on the surface of cancer cells, the method comprising administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having cancer characterized by having the population of cancer cells.

[0110] Furthermore, this specification provides a method for reducing the volume of a target tumor, wherein the tumor is characterized by having a population of cancer cells having TROP2 or an epitope of TROP2 presented on the surface of the cancer cells, and the method comprises administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a target identified as having a cancer characterized by having the population of cancer cells.

[0111] Furthermore, this specification provides a method for inducing cell death of cancer cells in a subject, wherein the cancer cells have a TROP2 or TROP2 epitope presented on their surface, and the method comprises administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having cancer characterized by having the population of cancer cells.

[0112] In some embodiments, cancer is a primary tumor. In some embodiments, cancer is a metastasis.

[0113] In some embodiments, the cancer is a non-T cell invasive tumor. In some embodiments, the cancer is a T cell invasive tumor.

[0114] Furthermore, this specification provides a method for reducing the risk of metastasis in a subject having cancer, or for reducing the risk of further metastasis, wherein the cancer is characterized by having a population of cancer cells having TROP2 or an epitope of TROP2 presented on the surface of the cancer cells, and the method comprises administering a therapeutically effective amount of one of the pharmaceutical compositions described herein or one of the ABPCs described herein to a subject identified as having cancer characterized by having the population of cancer cells.

[0115] In some embodiments, the cancer is a non-T cell invasive tumor. In some embodiments, the cancer is a T cell invasive tumor.

[0116] As used herein, the term “antigen-binding protein construct” means (i) a single polypeptide comprising at least one ABD (antigen-binding domain), or (ii) a complex of two or more polypeptides (e.g., identical or different polypeptides) that together form at least one ABD (antigen-binding domain). Non-limiting examples and embodiments of antigen-binding protein constructs are described herein. Further examples and embodiments of antigen-binding protein constructs are known in the art.

[0117] A "multispecific ABPC" is an ABPC comprising two or more different ABDs that collectively and specifically bind to two or more different epitopes. These two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or on different antigens (e.g., different proteins present on the surface of the same cell or on the surfaces of different cells). In some embodiments, the antigens are present on the surface of a cell. In some embodiments, a multispecific ABPC binds to two different epitopes (i.e., a "bispecific ABPC"). In some embodiments, a multispecific ABPC binds to three different epitopes (i.e., a "triplespecific ABPC"). In some embodiments, a multispecific ABPC binds to four different epitopes (i.e., a "quadrispecific ABPC"). In some embodiments, a multispecific ABPC binds to five different epitopes (i.e., a "quintuple-specific ABPC"). Each binding specificity may exist in any appropriate valency. Non-limiting examples of multispecific ABPCs are described herein.

[0118] An “antibody-binding protein” or “ABD” is one or more protein domains (for example, formed from amino acids derived from a single polypeptide, or from amino acids derived from two or more polypeptides (for example, identical or different polypeptides) that can specifically bind to one or more different antigens). In some embodiments, an ABD can bind to an antigen or epitope having specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, an ABD can be an antibody or a fragment thereof. In some embodiments, an ABD can include an alternative scaffold. Non-limiting examples of ABDs are described herein. Additional examples of ABDs are known in the art. In some embodiments, an ABD can bind to a single antigen.

[0119] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules, including one or more ABDs that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins, e.g., human IgG (e.g., human IgG1, human IgG2, human IgG3, human IgG4)), antibody fragments, and multispecific antibodies. An example of an ABD is an ABD formed by a VH-VL dimer. Additional examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0120] The term "endosomal / lysosomal pathway" refers to the network of endosomes (early endosomes, polyvesicles, late endosomes, and lysosomes) in the cytoplasm of mammalian cells, where molecules are selectively transported through cell-mediated internal migration processes, such as phagocytosis, microphagocytosis, receptor-mediated endocytosis, and / or phagocytosis.

[0121] Once endosomes from the endosomal / lysosomal pathway are purified or isolated, assays for target proteins (e.g., antigen-binding protein constructs described herein) can be performed using methods known in the art (ELISA, Western blotting, immunofluorescence, and immunoprecipitation followed by protein concentration assays) and can be used to determine the concentration or relative level of the target protein in the endosomes. Alternatively, endosomes from the endosomal / lysosomal pathway can be imaged by immunofluorescence microscopy using detectably labeled antibodies (e.g., fluorescently labeled, dye-labeled, or GFP-labeled antibodies, e.g., CellLight® Early Endosome-GFP) that specifically bind to characteristic proteins present in the endosomes (e.g., EEA1 in early endosomes), and fluorescently labeled antibodies that specifically bind to the target protein (e.g., ABPC), and the level of the target protein in the endosomes can be determined by quantifying the overlap of fluorescence emission from two different antibodies.

[0122] The term “endolysosome delivery” refers to the rate of accumulation over time or the total accumulation at a specific point in time of ABPCs (e.g., any of the ABPCs described herein) within the endosomal / lysosomal pathway in mammalian cells (e.g., any of the exemplary target mammalian cells described herein).

[0123] An exemplary method for calculating the increase in endolysosome delivery of pH-manipulated ABPC variants compared to the corresponding starting ABPC from cell fluorescence data is to subtract the average fluorescence intensity of the unbound IgG control from the average fluorescence intensity of the variant, and then measure the ratio obtained by dividing all of this by the value obtained by subtracting the average fluorescence intensity of the IgG control from the average fluorescence intensity of the corresponding starting ABPC of the variant.

[0124] Exemplary assays for measuring endolysosome delivery of any of the ABPCs described herein include labeling of the ABPC with a fluorescent dye, followed by incubation of the labeled ABPC with cells, and measurement of cell fluorescence as an indicator of ABPC endolysosome delivery (for example, outlined in Wustner, Traffic 7(6):699-715, 2006). Alternatively, any of the ABPCs described herein may be labeled using a pH-sensitive dye that preferentially fluoresces at acidic pH but not at neutral pH, and then incubated with cells to measure cell fluorescence as an indicator of ABPC delivery to the acidic endolysosomal compartment.

[0125] When the term “group” follows a noun, it refers to two or more of those specific nouns. For example, the phrase “group of cancer cells” means “two or more cancer cells.” Non-specific examples of cancer cells are given herein.

[0126] The term "cell proliferation inhibitory" refers to a direct or indirect reduction of cell proliferation (cell division) in vivo or in vitro in cells (e.g., cancer cells). When a drug is cell proliferation inhibitory, it can directly or indirectly cause cell cycle arrest in cells (e.g., cancer cells). In some cases, a cell proliferation inhibitory drug can reduce the number of cells in a population of cells in the S phase (compared to the number of cells in that population of cells in the S phase before contact with the drug). In some cases, a cell proliferation inhibitory drug can reduce the percentage of cells in the S phase by at least 20%, at least 40%, at least 60%, or at least 80% (compared to the percentage of cells in a population of cells in the S phase before contact with the drug).

[0127] The term "cytotoxic to cells" refers to the direct or indirect induction of cell death (e.g., necrosis or apoptosis) in the cells in question (e.g., mammalian cells, e.g., cancer cells).

[0128] "Affinity" refers to the sum of the non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects the 1:1 interaction between the ABD member and the antigen or epitope. The affinity of molecule X for its partner Y is given by the dissociation equilibrium constant (K). D Affinity can be expressed by the following: Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Further methods for determining affinity for ABD and its corresponding antigen or epitope are known in the art.

[0129] The term “epitope” refers to a portion of an antigen that is specifically bound by an ABD through a series of physical interactions between: (i) all monomers on the portion of the ABD that specifically binds to the antigen (e.g., individual amino acid residues, sugar side chains, and post-translational modified amino acid residues); and (ii) all monomers on the portion of the antigen that is specifically bound by an ABD (e.g., individual amino acid residues, sugar side chains, and post-translational modified amino acid residues). An epitope may consist, for example, surface-accessible amino acid residues, sugar side chains, phosphorylated amino acid residues, methylated amino acid residues, and / or acetylated amino acid residues, and may have specific three-dimensional structural properties as well as specific charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that, in the presence of a denaturing solvent, binding to the former may be lost, but binding to the latter is not. In some embodiments, an epitope is defined by a linear amino acid sequence of at least about 3–6 amino acids, or about 10–15 amino acids. In some embodiments, an epitope refers to a portion or part of a full-length protein defined by a three-dimensional structure (e.g., protein folding). In some embodiments, an epitope is defined by a discontinuous amino acid sequence joined together via protein folding. In some embodiments, an epitope is defined by a discontinuous amino acid sequence joined together by a quaternary structure (e.g., a cleavage formed by the interaction of two different polypeptide chains). The amino acid sequence between the residues defining the epitope may not be important for the three-dimensional structure of the epitope. Conformational epitopes can be determined and screened using assays that compare the binding of antigen-binding protein constructs to denatured versions of the antigen so that a linear epitope is generated. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding.

[0130] Methods for identifying epitopes to which ABD specifically binds are known in the art, and many of them are known in the art, such as structural analysis (e.g., X-ray crystallography, NMR, and / or electron microscopy) (e.g., for antigens and / or antigen-ABD complexes) and / or mutagenesis analysis in which mutants are measured by binding assays with binding partners (e.g., alanine scanning mutagenesis, glycine scanning mutagenesis, and homology scanning mutagenesis).

[0131] The term "paratope" refers to a portion of an ABD that specifically binds to an antigen through a series of physical interactions between: (i) all monomers on the portion of the ABD that specifically binds to the antigen (e.g., individual amino acid residues, sugar side chains, post-translational modified amino acid residues) and (ii) all monomers on the portion of the antigen that is specifically bound by the ABD (e.g., individual amino acid residues, sugar side chains, post-translational modified amino acid residues). A paratope may consist, for example, of surface-accessible amino acid residues and may have specific three-dimensional structural properties as well as specific charge properties. In some embodiments, a paratope refers to a portion or part of a full-length ABD defined by a three-dimensional structure (e.g., protein folding). In some embodiments, a paratope is defined by a discontinuous amino acid sequence brought together via protein folding. In some embodiments, an epitope is defined by a discontinuous amino acid sequence brought together by a quaternary structure (e.g., a rift formed by the interaction of two different polypeptide chains). The amino acid sequence between the residues that define the paratope may not be important for the three-dimensional structure of the paratope. A paratope may contain amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding.

[0132] Methods for identifying paratopes to which ABD specifically binds are known in the art, and many of them are known in the art, such as structural analysis (e.g., X-ray crystallography, NMR, and / or electron microscopy) (e.g., for ABD and / or ABD-antigen complexes) and / or mutation induction analysis (e.g., alanine scanning mutagenesis, glycine scanning mutagenesis, and homology scanning mutagenesis) that measure variants with binding partners.

[0133] The phrase "present on the surface of mammalian cells" means (1) an antigen that is physically bound to or at least partially incorporated into the plasma membrane of a mammalian cell (e.g., transmembrane proteins, peripheral membrane proteins, lipid-binding proteins (e.g., GPI anchors), N-myristolylated proteins, or S-palmitolylated proteins), or (2) an antigen that is stably bound to a homologous receptor that is physically attached to the plasma membrane of the cell (e.g., a ligand bound to that homologous receptor, which is physically attached to the plasma membrane). Non-limiting methods for determining the presence of antigens on the surface of mammalian cells include fluorescence-activated cell sorting (FACS), immunohistochemistry, cell fractionation assays, and Western blotting.

[0134] The terms “control ABPC” or “control antigen-binding protein construct” mean (i) an ABPC that can specifically bind to TROP2 or an epitope of TROP2 presented on the surface of a mammalian cell (e.g., a target mammalian cell) and one or both of the following are true: (a) the dissociation rate of the first antigen-binding domain (ABD) at pH approximately 4.0 to approximately 6.5 (e.g., any of the subranges of this range described herein) is similar to the dissociation rate of the first antigen-binding domain (ABD) at pH approximately 7.0 to approximately 8.0 (e.g., any of the subranges of this range described herein). (b) The dissociation rate of the first antigen-binding domain (ABD) is three times faster than (e.g., 2.8 times or less, 2.6 times or less, 2.5 times or less, 2.4 times or less, 2.2 times or less, 2.0 times or less, 1.8 times or less, 1.6 times or less, 1.5 times or less, 1.4 times or less, 1.2 times or less, 1.0 times or less, 0.8 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less) than the dissociation rate at (b) pH approximately 4.0 to approximately 6.5 (e.g., any of the subranges of this range described herein) D ) at a pH of approximately 7.0 to approximately 8.0 (for example, any of the lower ranges of this range as described herein) D This means ABPC; (ii) sacituzumab; (iii) datopotamab; (iv) hu7E6SVG, and / or (v) A1,2X4, which are three times or less greater than (for example, 2.8 times or less, 2.6 times or less, 2.5 times or less, 2.4 times or less, 2.2 times or less, 2.0 times or less, 1.8 times or less, 1.6 times or less, 1.5 times or less, 1.4 times or less, 1.2 times or less, 1.0 times or less, 0.8 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less).

[0135] The term "extracellular space" refers to the fluid outside the plasma membrane of a mammalian cell. When mammalian cells are in vitro, the extracellular space may be a liquid culture medium. When mammalian cells are in vivo, the extracellular space may be, for example, plasma, serum, blood, interstitial fluid, or lymph.

[0136] The term "endolysosome space" refers to the fluid enclosed by vesicles and organelles that constitute the endosomal / lysosomal pathway in mammalian cells.

[0137] The phrase “reduced level” or “decreased level” may be a reduction or decrease of at least 1% (e.g., at least 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99%) compared to a baseline level or value.

[0138] The term “cytotoxicity” refers to the ability of a drug (e.g., any of the ABPCs described herein) to directly or indirectly induce apoptosis and / or necrosis of mammalian cells (e.g., cancer cells), and is measured as a ratio over time or at relevant point in time. Methods for determining the cytotoxicity of cells are known in the art (e.g., trypan blue staining, microscopy, fluorescent co-cell sorting, and assays for detecting markers of apoptosis (e.g., annexin V)). In non-limiting examples, cytotoxicity can be measured, for example, by cytotoxicity at a single concentration of the drug, the IC50 of the drug (i.e., the concentration of the drug at which half of the maximum cytotoxicity is achieved), or the ratio of the drug's dissociation constant KD to mammalian cells divided by its IC50. In some non-limiting examples, the IC50 and / or KD ratios described herein are compared to those of a control ABPC (as defined herein) to optionally demonstrate that the ABPCs described herein have higher cytotoxicity than the control ABPC.

[0139] The term "toxin release" refers to the ability of mammalian cells (e.g., non-cancerous mammalian cells or cancerous cells) to internalize any of the ABPCs described herein conjugated with a toxin (e.g., either the ABPCs described herein or a control ABPC) (e.g., via phagocytosis and / or receptor-mediated endocytosis), and subsequently release the toxin conjugated to the ABPC, as measured over time or as a percentage at a specific point in time. Toxin release can be evaluated using a variety of different exemplary assays, e.g., ELISA, immunofluorescence, cell killing assays, cell cycle arrest assays, DNA damage assays, mass spectrometry, HPLC, and / or isotope-labeled toxins.

[0140] The terms "target cell," "target mammalian cell," or "mammalian target cell" refer to mammalian cells that have at least one TROP2 present on their surface. In some cases, mammalian target cells can be cancer cells. In some embodiments of target mammalian cells, the total is approximately the following (each ± ​​about 10%): 1-10E6, 1-9E6, 1-8E6, 1-7E6, 1-6E6, 1-5E6, 1-4E6, 1-3E6, 1-2E6, 1-1E6, 1-800,000, 1-600,000, 1-400,000, 1-200,000, 1-100,000, 1-80,000, 1-80,000, 1-75,000, 1-70,000, 1-65,000, 1-60,000, 1-55,000, 1-50,000, 1-45 It may have TROP2 present on the plasma membrane of target mammalian cells in any of the following ranges: 1,000, 1-40,000, 1-35,000, 1-30,000, 1-25,000, 1-20,000, 1-15,000, 1-10,000, 1-7,500, 1-5,000, 1-4,000, 1-3,000, 1-2,000, 1-1,000, 1-500, 1-100, 1-50, or 1-10, or any of the ranges of numbers listed in the paragraph numbers of U.S. Patent Application Publication No. 2022 / 0281984.

[0141] The term "antigen density" refers to the number of TROP2s present on the surface of target mammalian cells, or the average number of TROP2s on the surface of a population of a particular type of target mammalian cell. For example, it can be measured using a Quantibright bead kit or radiolabeling (e.g., BD Biosciences PE Phycoerythrin Fluorescence Quantitation Kit, catalog number 340495).

[0142] The phrase "histidine-substituted amino acid" refers to the substitution of a non-histidine amino acid residue in a reference polypeptide sequence with histidine. Non-limiting methods for substituting amino acid residues in a reference polypeptide with histidine are described herein. Additional methods for substituting amino acid residues in a reference polypeptide with histidine are known in the art.

[0143] The phrase "alanine-substituted amino acid" refers to the substitution of a histidine amino acid residue with alanine in a reference polypeptide sequence. Non-limiting methods for substituting histidine with alanine in a reference polypeptide are described herein. Further methods for substituting histidine with alanine in a reference polypeptide are known in the art.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Methods and materials described herein for use in the invention may also be used, as may other suitable methods and materials known in the art. Materials, methods, and examples are for illustrative purposes only and are not intended to limit the use of the invention. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, including definitions, this specification shall prevail.

[0145] Other features and advantages of the present invention will be apparent from the following detailed description and figures, as well as from the claims.

[0146] Many aspects of this disclosure can be better understood by referring to the following drawings. The elements in the drawings are not necessarily to exact scale, and instead the focus is on illustrating the principles of the present invention in an easily understandable way. [Brief explanation of the drawing]

[0147] [Figure 1A] Octet plots of sacituzumab and its heavy-chain (HC) and light-chain (LC) variants (MYT11566~MYT11621) are presented. 1A: The Y-axis is fixed; 1B: The Y-axis is automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 1B] Octet plots of sacituzumab and its heavy-chain (HC) and light-chain (LC) variants (MYT11566~MYT11621) are presented. 1A: The Y-axis is fixed; 1B: The Y-axis is automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 2A] Octet plots of sacituzumab and several LC variants (MYT11566, MYT11622-MYT11635), as well as datopotamab and its HC variants (MYT11622-MYT11676), are presented. 2A: The Y-axis is fixed; 2B: The Y-axis is automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 2B] Octet plots of sacituzumab and several LC variants (MYT11566, MYT11622-MYT11635), as well as datopotamab and its HC variants (MYT11622-MYT11676), are presented. 2A: The Y-axis is fixed; 2B: The Y-axis is automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 3A-1] Octet plots of additional HC and LC variants (MYT11635~MYT11755) are presented. 3A: X and Y axes are fixed; 3B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 3A-2] Same as above. [Figure 3B-1] Octet plots of additional HC and LC variants (MYT11635~MYT11755) are presented. 3A: X and Y axes are fixed; 3B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 3B-2] Same as above. [Figure 4A] Octet plots of additional HC and LC variants (MYT12389~MYT12425) are presented. 4A: X and Y axes are fixed; 4B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 4B] Octet plots of additional HC and LC variants (MYT12389~MYT12425) are presented. 4A: X and Y axes are fixed; 4B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 5A] Octet plots of additional HC and LC variants (MYT12405~MYT12473) are presented. 5A: X and Y axes are fixed; 5B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 5B] Octet plots of additional HC and LC variants (MYT12405~MYT12473) are presented. 5A: X and Y axes are fixed; 5B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 6A] Octet plots of additional HC and LC variants (MYT12385~MYT12409) are presented. 6A: X and Y axes are fixed; 6B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 6B] Octet plots of additional HC and LC variants (MYT12385~MYT12409) are presented. 6A: X and Y axes are fixed; 6B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 7A-1] Octet plots of additional HC and LC variants (MYT12296~MYT12465) are presented. 7A: X and Y axes are fixed; 7B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 7A-2] Same as above. [Figure 7B-1] Octet plots of additional HC and LC variants (MYT12296~MYT12465) are presented. 7A: X and Y axes are fixed; 7B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 7B-2] Same as above. [Figure 8A] Octet plots of additional HC and LC variants (MYT12294~MYT12456) are presented. 8A: X and Y axes are fixed; 8B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 8B]Octet plots of additional HC and LC variants (MYT12294~MYT12456) are presented. 8A: X and Y axes are fixed; 8B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 9] This graph shows the results of an internal distribution assay. It displays the median fluorescence intensity (MFI) for each IgG construct. [Figure 10] This is a graph showing the MFI of the IgG constructs presented. [Figure 11] This graph shows the cytotoxicity of NCI-H1975 cells as a function of foldover datopotamab (FoD) against the indicated antibody. [Figure 12] This graph shows the killing depth of NCI-H1975 cells as a function of FoD against the indicated antibody. [Figure 13A] Octet plots for variants MYT13160 to MYT13203 are presented. 13A: X and Y axes are fixed; 13B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 13B] Octet plots for variants MYT13160 to MYT13203 are presented. 13A: X and Y axes are fixed; 13B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 14A-1] Octet plots for variants MYT13204 to MYT13257 are presented. 14A: X and Y axes are fixed; 14B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 14A-2] Same as above. [Figure 14B-1]Octet plots for variants MYT13204 to MYT13257 are presented. 14A: X and Y axes are fixed; 14B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 14B-2] Same as above. [Figure 15A] Octet plots for variants MYT13258 to MYT13309 are presented. 15A: X and Y axes are fixed; 15B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 15B] Octet plots for variants MYT13258 to MYT13309 are presented. 15A: X and Y axes are fixed; 15B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 16A-1] Octet plots for variants MYT13366 to MYT13447 are presented. 16A: X and Y axes are fixed; 16B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 16A-2] Same as above. [Figure 16B-1] Octet plots for variants MYT13366 to MYT13447 are presented. 16A: X and Y axes are fixed; 16B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 16B-2] Same as above. [Figure 17A]Octet plots for linker-toxin-conjugated variants 112-DXd, 165-DXd, 166-DXd, 167-DXd, 168-DXd, 169-DXd, 170-DXd, 171-DXd, and 172-DXd are presented. 17A: X and Y axes are fixed; 17B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Figure 17B] Octet plots for linker-toxin-conjugated variants 112-DXd, 165-DXd, 166-DXd, 167-DXd, 168-DXd, 169-DXd, 170-DXd, 171-DXd, and 172-DXd are presented. 17A: X and Y axes are fixed; 17B: X and Y axes are automatically applied to maximize vertical separation. The gray and black lines correspond to pH 5.4 and pH 7.4, respectively. [Modes for carrying out the invention]

[0148] Additional benefits of this disclosure are partially described in the following description, partially evident from the description, or can be learned through the practice of this disclosure. The benefits of this disclosure will be realized and achieved through the elements and combinations specifically indicated in the attached claims. It should be understood that both the general description above and the detailed description below are illustrative and descriptive only and do not limit the claims of this disclosure.

[0149] This specification provides antigen-binding protein constructs (ABPCs) comprising a first ABD capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD at pH about 4.0 to about 6.5 is faster than the dissociation rate at pH about 7.0 to about 8.0, and / or (b) the dissociation constant (KD) of the first ABD at pH about 4.0 to about 6.5 is greater than the KD at pH about 7.0 to about 8.0. In some examples of these ABPCs, the ABPC is degraded in the target mammalian cell after internal translocation of the ABPC by the target mammalian cell. Any of the examples of ABPCs described herein may further include conjugated toxins, radioisotopes, drugs, or small molecules (e.g., fluorophores or dyes).

[0150] Furthermore, an antigen-binding protein construct (ABPC) comprising a first ABD capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, and a conjugated toxin, radioisotope, drug, or small molecule, wherein (a) the dissociation rate of the first ABD at pH approximately 4.0 to approximately 6.5 is faster than the dissociation rate at pH approximately 7.0 to approximately 8.0, and / or the dissociation constant (KD) of the first ABD at pH approximately 4.0 to approximately 6.5 is greater than the KD at pH approximately 7.0 to approximately 8.0. (b) an antigen-binding protein construct (ABPC) is provided, wherein a composition containing the ABPC results in one or more (e.g., two or three) of the following compared to a composition containing the same amount of control ABPC: increased toxin release in target mammalian cells (e.g., a detectable increase), increased killing of target mammalian cells (e.g., a detectable increase), and increased endolysosome delivery in target mammalian cells (e.g., a detectable increase).

[0151] In some examples, the first ABD comprises an HCVD of sacituzumab having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an LCVD of sacituzumab having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an HCVD of sacituzumab having one or more amino acids substituted with histidine and an LCVD of sacituzumab having one or more amino acids substituted with histidine. In some examples, the HCVD of sacituzumab comprises SEQ ID NO: 4. In some examples, the LCVD of sacituzumab comprises SEQ ID NO: 5. In each of the above examples, alanine may be substituted in place of histidine.

[0152] In some embodiments, the first ABD is an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, wherein one or more amino acid positions of SEQ ID NOs. 6, 7, and 8 are collectively substituted with histidine. In some embodiments, the first ABD is an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, wherein one or more amino acid positions of SEQ ID NOs. 9, 10, and 11 are collectively substituted with histidine. In some embodiments, the first ABD includes an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 6, 7, and 8, respectively, wherein one or more amino acid positions in SEQ ID NOs. 6, 7, and 8 are collectively substituted with histidine; and an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 9, 10, and 11, respectively, wherein one or more amino acid positions in SEQ ID NOs. 9, 10, and 11 are collectively substituted with histidine. In each of the above embodiments, alanine may be substituted in the location where histidine was already present.

[0153] In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, wherein the HCVD contains histidine at one or more amino acid positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine at one or more amino acid positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5. In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine at one or more amino acid positions selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 in SEQ ID NO: 4, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at one or more amino acid positions selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97 in SEQ ID NO: 5.

[0154] In some embodiments, the HCVD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and the HCVD contains histidine in one of the specific combinations of two or more amino acid positions in SEQ ID NO: 4 listed in Table 1.

[0155] [Table 1-1]

[0156] [Table 1-2]

[0157] [Table 1-3]

[0158] [Table 1-4]

[0159] [Table 1-5]

[0160] [Table 1-6]

[0161] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 5 listed in Table 2, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 5.

[0162] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and the LCVD contains alanine at position 91 of SEQ ID NO: 5.

[0163] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and an LCVD containing alanine at position 91 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and an HCVD containing histidine at one or more amino acid positions of SEQ ID NO: 4 selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108.

[0164] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 5, selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 5. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 5 selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 4 selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108.

[0165] In some embodiments, the first ABD is an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 5 selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 4 selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108, and / or the HCVD contains alanine at position 91.

[0166] [Table 2-1]

[0167] [Table 2-2]

[0168] [Table 2-3]

[0169] [Table 2-4]

[0170] [Table 2-5]

[0171] [Table 2-6]

[0172] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, wherein the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 5 listed in Table 2.

[0173] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 5 listed in Table 2, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, wherein the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0174] In some embodiments, the first ABD comprises an LCVD containing SEQ ID NO: 5, and an HCVD which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and which contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0175] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 28 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0176] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 29 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0177] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 30 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0178] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains alanine at position 91 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0179] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 31 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0180] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 32 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0181] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 33 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0182] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 50 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0183] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 51 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0184] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 52 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0185] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 53 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0186] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 89 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0187] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 92 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0188] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 93 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0189] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 94 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0190] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 95 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0191] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 96 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0192] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at position 97 of SEQ ID NO: 5, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 4 listed in Table 1.

[0193] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 4, and contains histidine at any one of the positions 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108 of SEQ ID NO: 4, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, and contains histidine at any of the specific combinations of two or more amino acid positions of SEQ ID NO: 5 listed in Table 2.

[0194] In some embodiments, the first ABD comprises an HCVD from one of the sequences shown in SEQ ID NOs. 4, SEQ ID NOs. 12-52, or one of the sequences shown in SEQ ID NOs. 313-350, and / or the first ABD comprises an LCVD from one of the sequences shown in SEQ ID NOs. 5, SEQ ID NOs. 53-79, or one of the sequences shown in SEQ ID NOs. 351-396.

[0195] In some embodiments of ABPC, the first ABD includes an LCVD containing one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53-79, or one of the sequences shown in SEQ ID NOs: 351-396, and an HCVD containing one of the sequences shown in SEQ ID NO: 4, SEQ ID NOs: 12-52, or one of the sequences shown in SEQ ID NOs: 313-350.

[0196] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 4, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0197] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 12, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0198] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 13, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0199] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 14, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0200] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 15, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0201] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 16, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0202] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 17, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0203] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 18, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0204] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 19, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0205] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 20, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0206] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 21, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0207] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 22, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0208] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 23, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0209] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 24, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0210] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 25, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0211] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 26, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0212] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 27, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0213] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 28, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0214] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 29, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0215] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 30, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0216] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 31, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0217] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 32, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0218] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 33, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0219] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 34, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0220] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 35, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0221] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 36, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0222] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 37, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0223] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 38, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0224] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 39, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0225] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 40, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0226] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 41, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0227] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 42, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0228] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 43, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0229] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 44, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0230] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 45, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0231] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 46, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0232] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 47, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0233] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 48, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0234] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 49, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0235] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 50, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0236] In some examples, the first ABD includes an HCVD that includes the sequence of SEQ ID NO: 51, and an LCVD that includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NOs: 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0237] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 52, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0238] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 313, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0239] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 314, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0240] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 315, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - �96.

[0241] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 316, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0242] In some examples, the first ABD includes a HCVD containing the sequence of SEQ ID NO: 317, and a LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79, or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0243] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 318, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0244] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 319, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0245] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 320, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0246] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 321, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0247] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 322, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0248] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 323, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0249] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 324, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0250] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 325, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0251] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 326, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0252] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 327, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0253] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 328, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0254] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 329, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0255] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 330 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0256] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 331 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0257] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 332 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0258] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 333 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0259] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 334 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0260] In some examples, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 335 and an LCVD containing one of the sequences shown in SEQ ID NOs: 5, 53 - 79 or one of the sequences shown in SEQ ID NOs: 351 - 396.

[0261] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 336, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0262] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 337, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0263] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 338, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0264] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 339, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0265] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 340, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0266] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 341, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0267] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 342, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0268] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 343, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0269] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 344, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0270] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 345, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0271] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 346, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0272] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 347, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0273] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 348, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0274] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 349, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0275] In some examples, the first ABD includes an HCVD containing the sequence of sequence number 350, and an LCVD containing one of the sequences shown in sequence number 5, sequence numbers 53-79, or one of the sequences shown in sequence numbers 351-396.

[0276] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 5, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0277] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 53, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0278] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 54, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0279] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 55, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0280] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 56, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0281] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 57, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0282] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 58, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0283] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 59, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0284] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 60, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0285] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 61, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0286] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 62, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0287] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 63, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0288] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 64, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0289] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 65, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0290] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 66, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0291] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 67, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0292] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 68, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0293] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 69, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0294] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 70, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0295] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 71, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0296] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 72, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0297] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 73, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0298] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 74, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0299] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 75, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0300] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 76, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0301] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 77, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0302] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 78, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0303] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 79, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0304] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 351, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0305] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 352, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0306] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 353, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0307] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 354, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0308] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 355, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0309] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 356, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0310] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 357, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0311] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 358, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0312] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 359, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0313] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 360, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0314] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 361, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0315] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 362, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0316] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 363, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0317] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 364, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0318] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 365, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0319] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 366, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0320] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 367, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0321] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 368, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0322] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 369, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0323] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 370, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0324] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 371, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0325] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 372, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0326] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 373, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0327] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 374, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0328] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 375, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0329] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 376, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0330] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 377, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0331] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 378, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0332] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 379, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0333] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 380, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0334] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 381, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0335] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 382, ​​and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0336] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 383, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0337] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 384, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0338] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 385, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0339] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 386, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0340] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 387, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0341] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 388, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0342] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 389, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0343] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 390, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0344] In some cases, the first ABD includes an LCVD containing the sequence of sequence number 391, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0345] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 392, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0346] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 393, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0347] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 394, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0348] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 395, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0349] In some examples, the first ABD includes an LCVD containing the sequence of sequence number 396, and an HCVD containing one of the sequences shown in sequence number 4, sequence numbers 12-52, or one of the sequences shown in sequence numbers 313-350.

[0350] In some examples, the first ABD comprises a datopotamab HCVD having one or more amino acids substituted with histidine. In some examples, the first ABD comprises a datopotamab LCVD having one or more amino acids substituted with histidine. In some examples, the first ABD comprises a datopotamab HCVD having one or more amino acids substituted with histidine and a datopotamab LCVD having one or more amino acids substituted with histidine. In some examples, the datopotamab HCVD comprises SEQ ID NO: 80. In some examples, the datopotamab LCVD comprises SEQ ID NO: 81. In each of the above examples, alanine may be substituted in place of histidine.

[0351] In some embodiments, the first ABD is an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, wherein one or more amino acid positions in SEQ ID NOs. 82, 83, and 84 are collectively substituted with histidine. In some embodiments, the first ABD is an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, wherein one or more amino acid positions in SEQ ID NOs. 85, 86, and 87 are collectively substituted with histidine. In some embodiments, the first ABD includes an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 82, 83, and 84, respectively, wherein one or more amino acid positions in SEQ ID NOs. 82, 83, and 84 are collectively substituted with histidine; and an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 85, 86, and 87, respectively, wherein one or more amino acid positions in SEQ ID NOs. 85, 86, and 87 are collectively substituted with histidine. In each of the above embodiments, alanine may be substituted in the location where histidine was already present.

[0352] In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 81 selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95. In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, and contains histidine at one or more amino acid positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108; and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, and contains histidine at one or more amino acid positions of SEQ ID NO: 81 selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95.

[0353] In some embodiments, the HCVD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, and the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0354] In some embodiments, the first ABD comprises an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 80, wherein the HCVD contains alanine at position 54 of SEQ ID NO: 80.

[0355] In some embodiments, the first ABD includes an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 80, and an HCVD containing alanine at position 54 of SEQ ID NO: 80, and an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 81, and an LCVD containing histidine at one or more amino acid positions of SEQ ID NO: 81 selected from SEQ ID NOs: 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95.

[0356] [Table 3-1]

[0357] [Table 3-2]

[0358] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 81 listed in Table 4, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 81.

[0359] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains alanine at position 91 of SEQ ID NO: 81.

[0360] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, comprising an LCVD containing alanine at position 91 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, comprising an HCVD containing histidine at one or more amino acid positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108.

[0361] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 81 selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95, and / or the LCVD contains alanine at position 91 of SEQ ID NO: 81. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, comprising histidine at one or more amino acid positions of SEQ ID NO: 81 selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95, and / or comprising alanine at position 91 of SEQ ID NO: 81; and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, comprising histidine at one or more amino acid positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108.

[0362] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, comprising histidine at one or more amino acid positions of SEQ ID NO: 81 selected from 26, 28, 29, 30, 31, 33, 34, 50, 51, 52, 53, 54, 89, 93, 94, and 95, and / or comprising alanine at position 91 of SEQ ID NO: 81; and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, comprising histidine at one or more amino acid positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108, and / or comprising alanine at position 54 of SEQ ID NO: 80.

[0363] [Table 4-1]

[0364] [Table 4-2]

[0365] [Table 4-3]

[0366] [Table 4-4]

[0367] [Table 4-5]

[0368] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, and contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, and contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 81 listed in Table 4.

[0369] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, comprising histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 81 listed in Table 4, and / or comprising alanine at position 91 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, comprising histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0370] In some embodiments, the first ABD comprises an LCVD containing SEQ ID NO: 81 and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, and which contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0371] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 26 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0372] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 28 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0373] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 29 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0374] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains alanine at position 91 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0375] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 30 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0376] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 31 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0377] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 33 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0378] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 34 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0379] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 50 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0380] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 51 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0381] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 52 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0382] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 53 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0383] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 54 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0384] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 89 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0385] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 93 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0386] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 94 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0387] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, wherein the LCVD contains histidine at position 95 of SEQ ID NO: 81, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 80 listed in Table 3.

[0388] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, and contains histidine at any one of the positions 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108 of SEQ ID NO: 80, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 81, and contains histidine at any of the specific combinations of two or more amino acid positions of SEQ ID NO: 81 listed in Table 4.

[0389] In some embodiments, the first ABD comprises an HCVD from one of the sequences shown in SEQ ID NOs. 80, 88-128, and one of the sequences shown in SEQ ID NOs. 397-406, and / or the first ABD comprises an LCVD from one of the sequences shown in SEQ ID NOs. 81, 129-155, or one of the sequences shown in SEQ ID NOs. 407-426.

[0390] In some embodiments of ABPC, the first ABD includes an LCVD containing one of the sequences shown in SEQ ID NOs. 81, SEQ ID NOs. 129-155, or one of the sequences shown in SEQ ID NOs. 407-426, and an HCVD containing one of the sequences shown in SEQ ID NOs. 80, SEQ ID NOs. 88-128, or one of the sequences shown in SEQ ID NOs. 397-406.

[0391] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 80, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0392] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 88, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0393] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 89, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0394] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 90, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0395] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 91, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0396] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 92, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0397] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 93, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0398] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 94, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0399] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 95, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0400] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 96, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0401] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 97, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0402] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 98, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0403] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 99, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0404] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 100, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0405] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 101, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0406] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 102, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0407] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 103, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0408] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 104, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0409] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 105, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0410] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 106, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0411] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 107, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0412] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 108, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0413] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 109, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0414] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 110, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0415] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 111, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0416] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 112, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0417] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 113, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0418] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 114, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0419] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 115, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0420] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 116, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0421] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 117, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0422] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 118, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0423] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 119, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0424] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 120, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0425] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 121, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0426] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 122, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0427] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 123, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0428] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 124, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0429] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 125, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0430] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 126, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0431] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 127, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0432] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 128, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0433] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 397, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0434] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 398, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0435] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 399, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0436] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 400, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0437] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 401, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0438] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 402, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0439] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 403, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0440] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 404, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0441] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 405, and an LCVD containing one of the sequences shown in SEQ ID NO: 81, SEQ ID NOs: 129-155, or one of the sequences shown in SEQ ID NOs: 407-426.

[0442] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 406, and an LCVD containing one of the sequences shown in sequence number 81, sequence numbers 129-155, or one of the sequences shown in sequence numbers 407-426.

[0443] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 81, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0444] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 129, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0445] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 130, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0446] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 131, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0447] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 132, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0448] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 133, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0449] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 134, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0450] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 135, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0451] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 136, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0452] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 137, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0453] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 138, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0454] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 139, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0455] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 140, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0456] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 141, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0457] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 142, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0458] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 143, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0459] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 144, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0460] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 145, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0461] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 146, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0462] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 147, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0463] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 148, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0464] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 149, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0465] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 150, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0466] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 151, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0467] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 152, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0468] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 153, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0469] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 154, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0470] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 155, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0471] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 407, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0472] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 408, and an HCVD containing one of the sequences shown in sequence numbers 80, 88-128, or 397-406.

[0473] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 409, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0474] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 410, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0475] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 411, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0476] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 412, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0477] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 413, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0478] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 414, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0479] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 415, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0480] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 416, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0481] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 417, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0482] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 418, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0483] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 419, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0484] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 420, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0485] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 421, and an HCVD containing one of the sequences shown in SEQ ID NO: 80, SEQ ID NOs: 88-128, or one of the sequences shown in SEQ ID NOs: 397-406.

[0486] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 422, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0487] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 423, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0488] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 424, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0489] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 425, and an HCVD containing one of the sequences shown in SEQ ID NOs: 80, 88-128, or 397-406.

[0490] In some embodiments, the first ABD includes an LCVD containing the sequence of sequence number 426, and an HCVD containing one of the sequences shown in sequence number 80, sequence numbers 88-128, or one of the sequences shown in sequence numbers 397-406.

[0491] In some examples, the first ABD comprises an HCVD of hu7E6SVG having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an LCVD of hu7E6SVG having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an HCVD of hu7E6SVG having one or more amino acids substituted with histidine and an LCVD of hu7E6SVG having one or more amino acids substituted with histidine. In some examples, the HCVD of hu7E6SVG includes SEQ ID NO: 156. In some examples, the LCVD of hu7E6SVG includes SEQ ID NO: 157. In each of the above examples, alanine may be substituted in place of histidine.

[0492] In some embodiments, the first ABD is an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, wherein one or more amino acid positions of SEQ ID NOs. 158, 159, and 160 are collectively substituted with histidine. In some embodiments, the first ABD is an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, wherein one or more amino acid positions of SEQ ID NOs. 161, 162, and 163 are collectively substituted with histidine. In some embodiments, the first ABD includes an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 158, 159, and 160, respectively, wherein one or more amino acid positions in SEQ ID NOs. 158, 159, and 160 are collectively substituted with histidine; and an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 161, 162, and 163, respectively, wherein one or more amino acid positions in SEQ ID NOs. 161, 162, and 163 are collectively substituted with histidine. In each of the above embodiments, alanine may be substituted in the location where histidine was already present.

[0493] In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 156 selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 157 selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101. In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, and contains histidine at one or more amino acid positions of SEQ ID NO: 156 selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106; and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, and contains histidine at one or more amino acid positions of SEQ ID NO: 157 selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101.

[0494] [Table 5-1]

[0495] [Table 5-2]

[0496] [Table 5-3]

[0497] In some embodiments, the HCVD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, and the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0498] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 157 listed in Table 6.

[0499] In some embodiments, the first ABD comprises an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 156, wherein the HCVD contains alanine at position 35 of SEQ ID NO: 156.

[0500] In some embodiments, the first ABD comprises an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 156, wherein the HCVD contains alanine at position 35 of SEQ ID NO: 156, and an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 157 selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101.

[0501] In some embodiments, the first ABD comprises an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 38 of SEQ ID NO: 157.

[0502] In some embodiments, the first ABD comprises an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 38 of SEQ ID NO: 157, and an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 156, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 156 selected from SEQ ID NOs: 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106.

[0503] In some embodiments, the first ABD comprises an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 94 of SEQ ID NO: 157.

[0504] In some embodiments, the first ABD comprises an LCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 94 of SEQ ID NO: 157, and an HCVD that is at least 90% (e.g., at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) homologous to SEQ ID NO: 156, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 156 selected from SEQ ID NOs: 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106.

[0505] [Table 6-1]

[0506] [Table 6-2]

[0507] [Table 6-3]

[0508] [Table 6-4]

[0509] [Table 6-5]

[0510] [Table 6-6]

[0511] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 157 listed in Table 6.

[0512] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 157 listed in Table 6, and / or the LCVD contains alanine at position 38 or 94 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0513] In some embodiments, the first ABD comprises an LCVD containing SEQ ID NO: 157 and an HCVD which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in one of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0514] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 25 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0515] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 32 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0516] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 34 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0517] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 38 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0518] In some embodiments, the first ABD includes an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains alanine at position 94 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0519] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 36 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0520] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 54 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0521] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 56 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0522] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 57 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0523] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 58 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0524] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 59 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0525] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 60 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0526] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 93 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0527] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 94 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0528] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 96 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0529] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 97 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0530] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 99 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0531] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 100 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0532] In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at position 101 of SEQ ID NO: 157, and an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine in any of the specific combinations of two or more amino acid positions of SEQ ID NO: 156 listed in Table 5.

[0533] In some embodiments, the first ABD includes an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 156, wherein the HCVD contains histidine at any one of the positions 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106 of SEQ ID NO: 156, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at any of the specific combinations of two or more amino acid positions of SEQ ID NO: 157 listed in Table 6.

[0534] In some embodiments, the first ABD includes an HCVD from one of the sequences shown in SEQ ID NOs. 156, SEQ ID NOs. 164-202, or one of the sequences shown in SEQ ID NOs. 427-457, and / or the first ABD includes an LCVD from one of the sequences shown in SEQ ID NOs. 157, SEQ ID NOs. 203-233, or one of the sequences shown in SEQ ID NOs. 458-492.

[0535] In some embodiments of ABPC, the first ABD includes an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0536] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 156, and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0537] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 164 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0538] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 165 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0539] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 166 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0540] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 167 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0541] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 168 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0542] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 169 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0543] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 170 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0544] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 171 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0545] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 172 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0546] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 173 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0547] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 174 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0548] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 175 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0549] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 176 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0550] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 177 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0551] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 178 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0552] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 179 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0553] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 180 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0554] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 181 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0555] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 182 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0556] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 183 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0557] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 184 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0558] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 185 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0559] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 186 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0560] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 187 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0561] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 188 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0562] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 189 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0563] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 190 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0564] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 191 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0565] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 192 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0566] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 193 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0567] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 194 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0568] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 195 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0569] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 196 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0570] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 197 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0571] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 198 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0572] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 199 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0573] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 200, and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0574] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 201 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0575] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 202 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0576] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 427 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0577] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 428 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0578] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 429 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0579] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 430 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0580] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 431 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0581] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 432 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0582] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 433 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0583] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 434 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0584] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 435 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0585] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 436 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0586] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 437 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0587] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 438 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0588] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 439 and an LCVD containing one of the sequences shown in sequence number 157, sequence numbers 203-233, or one of the sequences shown in sequence numbers 458-492.

[0589] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 440 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0590] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 441 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0591] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 442 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0592] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 443 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0593] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 444 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0594] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 445 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0595] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 446 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0596] In some embodiments, the first ABD includes an HCVD containing the sequence of sequence number 447 and an LCVD containing one of the sequences shown in sequence number 157, sequence numbers 203-233, or one of the sequences shown in sequence numbers 458-492.

[0597] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 448 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0598] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 449 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0599] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 450 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0600] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 451 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0601] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 452, and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0602] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 453 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0603] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 454 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0604] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 455, and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0605] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 456 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0606] In some embodiments, the first ABD includes an HCVD containing the sequence of SEQ ID NO: 457 and an LCVD containing one of the sequences shown in SEQ ID NO: 157, SEQ ID NOs: 203-233, or one of the sequences shown in SEQ ID NOs: 458-492.

[0607] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 157, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0608] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 203 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0609] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 204 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0610] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 205 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0611] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 206 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0612] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 207 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0613] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 208 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0614] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 209 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0615] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 210 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0616] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 211 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0617] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 212, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0618] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 213 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0619] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 214 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0620] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 215 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0621] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 216, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0622] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 217 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0623] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 218 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0624] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 219 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0625] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 220 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0626] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 221 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0627] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 222, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0628] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 223 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0629] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 224 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0630] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 225 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0631] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 226 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0632] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 227 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0633] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 228 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0634] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 229 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0635] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 230 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0636] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 231 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0637] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 232, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0638] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 233 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0639] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 458, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0640] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 459 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0641] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 460 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0642] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 461 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0643] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 462 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0644] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 463 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0645] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 464 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0646] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 465 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0647] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 466, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0648] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 467 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0649] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 468, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0650] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 469 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0651] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 470 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0652] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 471 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0653] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 472 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0654] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 473 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0655] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 474 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0656] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 475 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0657] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 476 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0658] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 477 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0659] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 478 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0660] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 479 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0661] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 480 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0662] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 481 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0663] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 482 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0664] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 483 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0665] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 484 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0666] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 485 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0667] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 486 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0668] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 487 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0669] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 488, and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0670] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 489 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0671] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 490 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0672] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 491 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0673] In some embodiments, the first ABD includes an LCVD containing the sequence of SEQ ID NO: 492 and an HCVD containing one of the sequences shown in SEQ ID NO: 156, SEQ ID NOs: 164-202, or one of the sequences shown in SEQ ID NOs: 427-457.

[0674] In some examples, the first ABD comprises an A1,2X4 HCVD having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an A1,2X4 LCVD having one or more amino acids substituted with histidine. In some examples, the first ABD comprises an A1,2X4 HCVD having one or more amino acids substituted with histidine and an A1,2X4 LCVD having one or more amino acids substituted with histidine. In some examples, the A1,2X4 HCVD comprises SEQ ID NO: 234. In some examples, the A1,2X4 LCVD comprises SEQ ID NO: 235. In each of the above examples, alanine may be substituted in place of histidine.

[0675] In some embodiments, the first ABD is an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, wherein one or more amino acid positions of SEQ ID NOs. 236, 237, and 238 are collectively substituted with histidine. In some embodiments, the first ABD is an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, wherein one or more amino acid positions of SEQ ID NOs. 239, 240, and 241 are collectively substituted with histidine. In some embodiments, the first ABD includes an HCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 236, 237, and 238, respectively, wherein one or more amino acid positions in SEQ ID NOs. 236, 237, and 238 are collectively substituted with histidine; and an LCVD containing CDR1, CDR2, and CDR3 of SEQ ID NOs. 239, 240, and 241, respectively, wherein one or more amino acid positions in SEQ ID NOs. 239, 240, and 241 are collectively substituted with histidine. In each of the above embodiments, alanine may be substituted in the location where histidine was already present.

[0676] In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 234. In some embodiments, the first ABD comprises an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 235. In some embodiments, the first ABD comprises an HCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 234, wherein the HCVD contains histidine at one or more amino acid positions of SEQ ID NO: 234, and an LCVD that is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, wherein the LCVD contains histidine at one or more amino acid positions of SEQ ID NO: 235. In each of the above embodiments, alanine may be substituted at the location where histidine was already present.

[0677] Furthermore, provided herein are pharmaceutical compositions comprising any of the ABPCs described herein. Also provided herein are methods for treating a subject in need of such treatment, comprising administering a therapeutically effective amount of any of the ABPCs described herein to that subject.

[0678] In some embodiments, a composition comprising ABPC (e.g., any of the ABPCs described herein) may provide an increase (e.g., a detectable increase) in toxin release in target mammalian cells (e.g., any of the target mammalian cells described herein) compared to a composition comprising the same amount of control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., within the range of a percentage increase or a percentage increase as enumerated on pages 389-404 of WO 2021 / 022039 (or as enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984)).

[0679] In some embodiments, a composition comprising ABPC (e.g., any of the ABPCs described herein) may provide an increase (e.g., a detectable increase) in toxin release in target mammalian cells (e.g., any of the exemplary control ABPCs described herein) compared to a composition comprising the same amount of control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., within the range of a multiplicative increase or multiplicative increase as enumerated on pages 404-420 of WO 2021 / 022039 (or as enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984)).

[0680] In some embodiments, a composition containing ABPC (e.g., any of the ABPCs described herein) shows an increased (e.g., a detectable increase) in killing target mammalian cells (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9%) compared to a composition containing the same amount of control ABPC. We can offer increases of 5%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1,000%, 2,000%, 3,000%, 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, or 10,000%, or an increase of approximately 1% to 10,000% (for example, either in the lower range).

[0681] In some examples, a composition containing ABPC (e.g., any of the ABPCs described herein) showed an increased killing of target mammalian cells (e.g., a detectable increase) (e.g., at least 0.1-, 0.2-, 0.3-, 0.4-, 0.5-, 0.6-, 0.7-, 0.8-, 0.9-, 1.0-, 1.2-, 1.4-, 1.5-, 1.6-, 1.8-, 2.0-, 2.2-, 2.4-, 2.5) compared to a composition containing the same amount of control ABPC. It can provide an increase of -, 2.6-, 2.8-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 5.5-, 6.0-, 6.5-, 7.0-, 7.5-, 8.0-, 8.5-, 9.0-, 9.5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 80-, 85-, 90-, 95 times, or at least a 100-fold increase, or an increase of 0.1 to 100 times.

[0682] In some embodiments, a composition containing any of the ABPCs described herein (for example, upon contact with target mammalian cells presenting TROP2 on their surface) yields an IC of the same amount as a composition containing the same amount of control ABPC. 50 Compared to (for example, a decrease of ≥ 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least a decrease of 99%, a decrease of approximately 1% to 99%, or any of the lower ranges of this range as described herein) IC 50 This results in (the killing of target mammalian cells).

[0683] In some embodiments, compositions comprising any of the ABPCs described herein (for example, upon contact with target mammalian cells presenting TROP2 on their surface) show, for example, a difference in K on target mammalian cells presenting TROP2 on their surface at a neutral pH compared to a control ABPC. D IC at neutral pH on the same target cells 50An increase in the ratio to (for example, an increase of at least 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 times, or at least a 100 times increase, or an increase of 0.1 to 500 times (or any of the lower ranges of this range described herein) can be provided.

[0684] In some examples, compositions containing ABPC (e.g., any of the ABPCs described herein) showed increased endolysosome delivery in target mammalian cells (e.g., a detectable increase) (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1%) compared to compositions containing the same amount of control ABPC. It may provide increases of 20%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1,000%, 2,000%, 3,000%, 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, or at least 10,000% or about 1% to 10,000% (for example, or any of the lower ranges of this range as described herein).

[0685] In some examples, compositions containing ABPC showed increased endolysosome delivery in target mammalian cells (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3. It can provide increases of 0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 times or more, or at least a 100-fold increase, or an increase of approximately 0.1 to 100 times.

[0686] In any of the ABPC examples described herein, the target mammalian cells either do not express the FcRn receptor or express a lower level (e.g., a detectably low level) of the FcRn receptor (e.g., at least 1% reduction, at least 2%, 5%, 10% reduction, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% reduction, or at least 99% reduction) compared to FcRn-expressing control cells (e.g., HUVEC-ThermoFisher #C0035C). In some examples, the target mammalian cells are cancer cells. In some examples, the ABPC is cytotoxic or inhibitory to the target mammalian cells.

[0687] In some embodiments, a composition containing any of the ABPCs described herein (e.g., when administered to a subject) does not result in a significant reduction in the level of TROP2 presented on the surface of target cells compared to a composition containing the same amount of control ABPC (e.g., any of the control ABPCs described herein) (e.g., a 1% reduction to about 99% reduction, or any of the lower ranges described herein). In some embodiments, the composition does not result in a detectable reduction in the level of TROP2 presented on the surface of target mammalian cells.

[0688] In some embodiments, ABPC is cross-reactive with non-human primate TROP2 and human TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, and one or both of rat TROP2 and mouse TROP2. In some embodiments, ABPC is cross-reactive with non-human primate TROP2, human TROP2, rat TROP2, and mouse TROP2. In some embodiments, ABPC is cross-reactive with mouse TROP2 and rat TROP2. In some embodiments, ABD binds to TROP2 epitopes present on the surface of cells derived from Old World monkeys.

[0689] Some embodiments of any ABPC described herein may further include a second ABD (e.g., any of the exemplary ABDs described herein). Non-limiting embodiments of these methods are described below and may be used in any combination without limitation. Further embodiments of these methods are known in the art.

[0690] TROP2 or TROP2 epitope Several TROP2-conjugated monoclonal antibodies have been documented in the literature and can be used as templates for manipulating pH-dependent binding. The amino acid sequence of mature human TROP2 can be found in SEQ ID NO: 2. The amino acid sequence of the extracellular domain of TROP2 can be found in SEQ ID NO: 3.

[0691] Antigen-binding protein constructs Any of the antigen-binding protein constructs (ABPCs) described herein may be a single polypeptide or may comprise two, three, four, five, six, seven, eight, nine, or ten (identical or different) polypeptides. In some embodiments where the ABPC is a single polypeptide, the ABPC may comprise one ABD or two ABDs. In some embodiments where the ABPC is a single polypeptide and comprises two ABDs, the first and second ABDs may be identical or different from each other (and may also be able to specifically bind to the same or different antigens or epitopes).

[0692] In some embodiments where ABPC is a single polypeptide, the first ABD and the second ABD (if any) may be independently selected from the group consisting of VH domains, VHH domains, VNAR domains, and scFv, respectively. In some embodiments where ABPC is a single polypeptide, the antigen-binding protein construct may be BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scdiabody, scdiabody-CH3, scFv-CH-CL-scFv, HSAbody, scdiabody-HAS, tandem-scFv, adonectin, DARPin, fibronectin, DEP conjugate, PROTAB, or PROTAC. Additional examples of ABDs that may be used when ABPC is a single polypeptide are known in the art.

[0693] V H The H domain is a single monomeric variable antibody domain that can be found in camelids. NAR The domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. H H domain and V NARNon-limiting aspects of the domain include, for example, Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014;Muyldermans, J. Biotechnol. 74:277-302, 2001;Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001;Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013;Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. This is described in 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0694] In some embodiments where ABPC is a single polypeptide and includes two ABDs, both the first and second ABDs may be VHH domains, or at least one ABD may be a VHH domain. NAR It is a domain, or at least one ABD is V NAR It is a domain. In some embodiments where ABPC is a single polypeptide, the first ABD is an scFv domain. In some embodiments where ABPC is a single polypeptide and includes two ABDs, both the first and second ABDs may be scFv domains, or at least one ABD may be an scFv domain.

[0695] In some embodiments, the ABPC may contain two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides). In some embodiments where the ABPC contains two or more polypeptides, two, three, four, five, or six of the polypeptides may be identical.

[0696] In some embodiments, where the ABPC comprises two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides), two or more polypeptides of the ABPC associate (non-covalently) to form one or more ABDs, e.g., antigen-binding fragments of antibodies (e.g., any of the antigen-binding fragments of antibodies described herein), VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DA F (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, VHH-Fc, Tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, Dock and Lock Antibody antigen-binding fragments can form (lock), ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adonectin, DARPin, Fibronectin, DEP conjugate, PROTAB, or PROTAC. See, for example, Spiess et al., Mol. Immunol. 67:95-106, 2015, the entire description of these elements is incorporated herein. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments.Further examples of antibody antigen-binding fragments include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA), antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0697] The "Fv" fragment contains a non-covalent dimer of one HCVD and one LCVD.

[0698] The "Fab" fragment, in addition to the heavy chain variable domain and LCVD of the Fv fragment, also contains the light chain constant domain and the first heavy chain constant domain (C H1 ) includes.

[0699] The "F(ab')2" fragment contains two Fab fragments joined near the hinge region by disulfide bonds.

[0700] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to the polyvalent and polyspecific binding proteins described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patents No. 7,612,181; No. 8,258,268; No. 8,586,714; No. 8,716,450; No. 8,722,855; No. 8,735,546; and No. 8,822,645, each of which is incorporated into the whole by reference.

[0701] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0702] Further embodiments of ABPC are known in the art.

[0703] antigen-binding domain In any of the embodiments of the antigen-binding protein constructs (ABPCs) described herein, the first ABD (and, if applicable, the second ABD) is present at a pH of about 4.0 to about 6.5 (e.g., about 4.0 to about 6.4, or any of the pH ranges enumerated on pages 428-430 of WO2021 / 022039, which is incorporated herein by reference in its entirety (or enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984)). The dissociation rate of the second ABD is approximately 7.0 to 8.0 (for example, approximately 7.0 to 7.9, 7.0 to 7.8, 7.0 to 7.7, 7.0 to 7.6, 7.0 to 7.5, 7.0 to 7.4, 7.0 to 7.3, 7.0 to 7.2, 7.0 to 7.1, 7.1 to 8.0, 7.1 to 7.9, 7.1 to 7.8, 7.1 to 7.7, 7.1 to 7.6, 7.1 to 7.5, 7.1 to 7.4, 7.1 to 7.3, 7.1 to 7.2, 7.2 to 8.0, 7.2 to 7.9, 7.2 to 7.8, 7.2 to 7.7, 7 0.2~7.6, 7.2~7.5, 7.2~7.4, 7.2~7.3, 7.3~8.0, 7.3~7.9, 7.3~7.8, 7.3~7.7, 7.3~7.6, 7.3~7.5, 7.3~7.4, 7.4~8.0, 7.4~7.9, 7.4~7.8, 7.4~7.7, 7.4~7.6, 7.4~7.5, 7.5~8.0, 7.5~7.9, 7.5~7.8, 7.5~7.7, 7.5~7.6, 7.5~8.0, 7.6~7.9, 7.6~7.8, 7.6~7 Faster than the dissociation rate at pH 0.7, 7.7–8.0, 7.7–7.9, 7.7–7.8, 7.8–8.0, 7.8–7.9, or approximately 7.9–8.0 (e.g., at least 5% faster, or by the percentages listed on pages 430–447 of WO2021 / 022039 (or in the corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entirety, or by the range of those percentages).

[0704] In some embodiments of the antigen-binding protein constructs (ABPCs) described herein, the dissociation constant (K) of the first ABD (and, optionally, the second ABD, if present) is set at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein). D) is K at a pH of approximately 7.0 to approximately 8.0 (for example, any of the lower ranges of this range as described herein). D Larger than (e.g., detectably large) (e.g., at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 1,000, 2,000, 3 ,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or at least about 10,000% larger, or about 5% to about 10,000% larger, or larger than those listed on pages 448-466 of WO2021 / 022039 (or listed in the corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entirety. D (Any of the percentage ranges).

[0705] In some embodiments of the antigen-binding protein constructs (ABPCs) described herein, the dissociation rate of the first ABD (and, optionally, the second ABD, if present) at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein) is faster than the dissociation rate at a pH of about 7.0 to about 8.0 (e.g., or any of the subranges of this range) (e.g., at least 0.2 times faster, or by a factor of 1 / 2000 or a factor of 10000 faster, or by a factor of 10000 faster, or by a factor of 10000 faster, as enumerated on pages 466-468 of WO2021 / 022039, which is incorporated herein in its entirety by reference (or enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984)).

[0706] In some embodiments of the antigen-binding protein constructs (ABPCs) described herein, the dissociation constant (K) of the first ABD (and, optionally, the second ABD, if present) at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein) is used. D) is K at a pH of approximately 7.0 to approximately 8.0 (for example, any of the lower ranges of this range as described herein). D Larger than (e.g., detectably large) (e.g., at least 0.2 times larger, or by a multiple or range of multiples listed on pages 468–470 of WO2021 / 022039, which is incorporated herein in its entirety by reference (or listed in the corresponding U.S. Patent Application No. 2022 / 0281984)).

[0707] In some embodiments of ABPCs including a first ABD and a second ABD, the first and second ABDs are identical or at least 80% homologous to each other in their amino acid sequences (e.g., ≥82%, ≥84%, ≥86%, ≥88%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homologous). In some embodiments, ABPCs including a first ABD and a second ABD, the first ABD and the second ABD have sequences that are less than 80% identical to each other (e.g., <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, or <5% homologous). In some embodiments of ABPC, which include first and second ABDs, the first and second ABDs bind to two different epitopes (e.g., two different epitopes on TROP2, or a first ABD that specifically binds to TROP2 and a second ABD that binds to an antigen other than TROP2).

[0708] In some embodiments of the ABPC described herein, the first ABD (and, optionally, the second ABD, if present) has a pH of about 7.0 to 8.0 (e.g., any of the lower ranges of this range described herein). D This range is between approximately 1 pM and 5 μM (for example, 1 pM to 2 μM, or as enumerated on pages 471-492 of WO2021 / 022039, which is incorporated herein by reference in its entirety (or as enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984) KD (It is one of the ranges.)

[0709] In some embodiments of the ABPC described herein, the K of the first ABD (and, optionally, the second ABD, if present) at a pH of about 4.0 to 6.5 (e.g., any of the lower ranges of this range described herein) D This includes K molecules greater than 1 nM (for example, between approximately 1 nM and 1 mM, or those listed on pages 492-496 of WO2021 / 022039, which is incorporated herein by reference in whole (or listed in the corresponding U.S. Patent Application No. 2022 / 0281984) D It could be any of the ranges.

[0710] Various different methods known in the art may be used to construct any antigen-binding protein construct described herein. D It can be used to determine values ​​(e.g., electrophoretic mobility assays, filter binding assays, surface plasmon resonance assays, biomolecular binding kinetic assays, in vitro binding assays of antigen-expressing cells, etc.).

[0711] In some embodiments, the in vivo half-life of ABPC is reduced (e.g., a detectable reduction) (e.g., a reduction of at least 1%, or within the range of a percentage reduction or percentage reduction enumerated on pages 496-500 of WO2021 / 022039, which is incorporated herein by reference in its entirety (or enumerated in the corresponding U.S. Patent Application No. 2022 / 0281984)) compared to the half-life of a control ABPC (e.g., any of the exemplary control ABPCs described herein). Conversely, in some embodiments, the in vivo half-life of ABPC may be increased compared to the half-life of a control ABPC.

[0712] Conjugate In some embodiments, the ABPCs provided herein may be conjugated with drugs (e.g., chemotherapeutic agents, small molecules), toxins, or radioisotopes. Non-limiting examples of drugs, toxins, and radioisotopes (e.g., useful in the treatment of cancer) are known in the Art.

[0713] In some embodiments, at least one polypeptide of any of the ABPCs described herein is conjugated to a toxin, radioisotope, or drug via a cleavable linker. In some embodiments, the cleavable linker includes a protease cleavage site. In some embodiments, the cleavable linker is cleaved on the ABPC upon transport to lysosomes or late endosomes by target mammalian cells. In some embodiments, cleavage of the linker functionally activates the drug or toxin.

[0714] In some embodiments, at least one polypeptide of any of the ABPCs described herein is conjugated to a toxin, radioisotope, or drug via a non-cleavable linker. In some embodiments, the conjugated toxin, radioisotope, or drug is released during the lysosome and / or late endosome degradation of the ABPC.

[0715] Non-limiting examples of cleavable linkers include hydrazone linkers, peptide linkers, disulfide linkers, and thioether linkers. See, for example, Carter et al., Cancer J. 14(3):154-169, 2008; Sanderson et al., Clin. Cancer Res. 11(2 Pt1):843-852, 2005; Chari et al., Acc. Chem. Res. 41(1):98-107, 2008; Oflazoglu et al., Clin. Cancer Res. 14(19): 6171-6180, 2008; and Lu et al., Int. J. Mol. Sci. 17(4): 561, 2016.

[0716] Non-limiting examples of non-cleavable linkers include maleimide alkane linkers and maleimide cyclohexane linkers (MMCs) (see, e.g., McCombs et al., AAPS J. 17(2):339-351, 2015).

[0717] In some embodiments, any of the ABPCs described herein are cytotoxic or inhibitory to target mammalian cells.

[0718] In some embodiments, the antibodies provided herein may contain one or more amino acid substitutions that provide a conjugate site (e.g., to be conjugated with a drug, toxin, or radioisotope). In some embodiments, the antibodies provided herein may have one conjugate site. In some embodiments, the antibodies described herein may have two conjugate sites. In some embodiments, the antibodies provided herein may have three or more conjugate sites.

[0719] Natural cysteine ​​amino acids can also provide conjugates. In some embodiments, the antibodies provided herein may have a drug, toxin, or radioisotope conjugated at one or more naturally derived conjugate sites. In some embodiments, the antibodies provided herein may have a drug, toxin, or radioisotope conjugated at one or more naturally derived conjugate sites. In some embodiments, the antibodies provided herein may have a drug, toxin, or radioisotope conjugated at one or more naturally derived conjugate sites and one or more manipulated conjugate sites.

[0720] Conjugates via engineered cysteine ​​are achieved by methods known in the art. Briefly, engineered cysteine-containing antibodies are prepared for conjugate by treatment with a reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or 2-mercaptoethanol (BME). In this reduction reaction, the reducing agent breaks the interchain disulfides, including the disulfide bonds in the antibody, and removes the disulfide cap from the engineered cysteine. An optional re-oxidation step, achieved by exposure of the solution to air or an oxidizing agent, such as dehydroascorbic acid, allows for the reformation of the interchain disulfide bonds, leaving engineered cysteine ​​with thioate-reactive groups. Conjugates with maleimide functional groups on the linker-payload maleimide-vc-MMAE are achieved by reaction with the payload in a buffer solution containing a cosolvent, such as ethanol, dimethylacetamide (DMA), or dimethyl sulfoxide (DMSO). The crude conjugated antibody solution is purified by size exclusion chromatography or a selective filtration method, such as tangential flow filtration. In this step, residual unreacted payload, reducing agent, and oxidizing agent are removed from the reaction mixture, and the conjugated ADC product may be transferred to a desired formulation buffer.

[0721] Conjugation via hinge cysteine ​​is achieved in a similar manner using antibodies with or without additionally manipulated cysteine ​​conjugate sites. Briefly, this antibody is prepared for conjugation by treatment with a reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT). The reducing strength and concentration of the reducing agent are selected so that some or all of the interc...

Claims

1. A first antigen-binding domain (ABD) capable of specifically binding to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, wherein the dissociation rate of the first ABD at pH approximately 4.0 to approximately 6.5 is faster than the dissociation rate at pH approximately 7.0 to approximately 8.0, or the K of the first ABD at pH approximately 4.0 to approximately 6.5 Ｄ However, K at pH approximately 7.0 to 8.0 Ｄ A larger antigen-binding protein construct (ABPC).

2. The ABPC according to claim 1, wherein the first ABD comprises one heavy chain variable domain (HCD) from among (a) sacituzumab, (b) datopotamab, (c) hu7E6SVG, and (d) A1,2X4 HCVD, and / or one light chain variable domain (LCD) from among (a) sacituzumab, (b) datopotamab, (c) hu7E6SVG, and (d) A1,2X4 LCVD, and each HCVD or each LCVD has one or more amino acids substituted with histidine.

3. The ABPC according to claim 2, wherein the HCVD comprises one of the following: (a1) HCVD of sacituzumab including SEQ ID NO: 4, (b1) HCVD of datopotamab including SEQ ID NO: 80, (c1) HCVD of hu7E6SVG including SEQ ID NO: 156, and (d1) HCVD of A1,2X4 including SEQ ID NO: 234, and the corresponding LCVD of one of the following: (a2) LCVD of sacituzumab including SEQ ID NO: 5, (b2) LCVD of datopotamab including SEQ ID NO: 81, (c2) LCVD of hu7E6SVG including SEQ ID NO: 157, and (d2) LCVD of A1,2X4 including SEQ ID NO: 235, and each HCVD and / or each LCVD has one or more histidine substitutions.

4. The ABPC according to claim 3, wherein the first ABD comprises (a) to (c): (a1) an HCVD comprising one of the CDRs 6, 7, and 8 from sequence numbers 6, 7, and 8; (b1) sequence numbers 82, 83, and 84; (c1) sequence numbers 158, 159, and 160; and (d1) one CDR 1, 2, and 3 from sequence numbers 236, 237, and 238; and the corresponding LCVD comprising one of the CDRs 1, 2, and 3 from sequence numbers 9, 10, and 11; (b2) sequence numbers 85, 86, and 87; (c2) sequence numbers 161, 162, and 163; and (d2) one CDR 1, 2, and 3 from sequence numbers 239, 240, and 241, wherein each set of three CDRs collectively has one or more amino acid positions substituted with histidine.

5. (a) to (c): (a) HCVD which is at least 90%, 95%, or 98% identical to SEQ ID NO: 4, wherein the HCVD contains histidine at one or more positions of SEQ ID NO: 4 selected from 29, 32, 33, 50, 52, 53, 60, 61, 62, 63, 64, 65, 66, 99, 101, 104, 105, and 108. (b) HCVD which is at least 90%, 95%, or 98% identical to SEQ ID NO: 80, wherein the HCVD contains histidine at one or more positions of SEQ ID NO: 80 selected from 29, 53, 55, 64, 65, 98, 101, 104, 105, 107, and 108, and (c) The ABPC according to any one of claims 2 to 4, wherein the HCVD is at least 90%, 95%, or 98% identical to SEQ ID NO: 156, and the HCVD comprises one HCVD containing histidine at one or more positions of SEQ ID NO: 156 selected from 26, 29, 30, 33, 34, 51, 54, 56, 57, 59, 102, 103, and 106.

6. (a) to (d): (a) LCVD which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 5, wherein the LCVD contains histidine at one or more positions of SEQ ID NO: 5 selected from 28, 29, 30, 31, 32, 33, 50, 51, 52, 53, 89, 92, 93, 94, 95, 96, and 97. (b) LCVD which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 80, wherein the LCVD contains histidine at one or more positions of SEQ ID NO: 80 selected from 26, 28-31, 33, 34, 50-54, 89, 93, 94, and 95, (c) LCVD which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 157, wherein the LCVD contains histidine at one or more positions of SEQ ID NO: 157 selected from 25, 32, 34, 36, 54, 56, 57, 58, 59, 60, 93, 96, 97, 99, 100, and 101, and (d) The ABPC according to any one of claims 2 to 5, comprising one LCVD, which is at least 90%, 95%, or 98% homologous to SEQ ID NO: 235, and which contains histidine at one or more positions in SEQ ID NO:

235.

7. The ABPC according to claim 5 or 6, wherein the HCVD and / or LCVD comprises histidine at two or more positions in the defined sequence number.

8. The first ABD mentioned above is sequence number 4, one of the sequences sequence numbers 12-52, sequence number 80, sequence number The HCVD includes one of the sequences 88-128, one of the sequences 156, 164-202, one of the sequences shown in sequence number 234, sequence numbers 242-280, or one of the sequences shown in sequence numbers 318, 320, 322, 323, 324, 325, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, 348, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445, 446, 447, 449, 451, 454, 456, and 457, and / or the first ABD includes sequence number 5, sequence number One of the sequences shown in 53-79, sequence number 81, one of the sequences shown in sequence numbers 129-155, sequence number 157, one of the sequences shown in sequence numbers 203-233, sequence number 235, one of the sequences shown in sequence numbers 281-312, or sequence numbers 351, 352, 353, 354, 355, 359, 360, 361, 362, 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 3 The ABPC according to claim 1, comprising an LCVD containing one of the sequences shown in 88, 389, 392, 393, 394, 395, 396, 411, 412, 417, 418, 423, 459, 460, 462, 464, 465, 466, 467, 468, 469, 470, 473, 474, 485, 488, 489, and 491.

9. The first ABD mentioned above is (a) to (c): (a) HCVD of one of the sequences shown in SEQ ID NOs. 4, SEQ ID NOs. 12-52, or one of the sequences shown in SEQ ID NOs. 313-350, and / or LCVD of one of the sequences shown in SEQ ID NOs. 5, SEQ ID NOs. 53-79, or one of the sequences shown in SEQ ID NOs. 351-396 (the first ABD is (i) the HCVD of SEQ ID NOs. 4 and 5, (ii) the HCVD of SEQ ID NOs. 4, and SEQ ID NOs. 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 71, 74, 75, 76, 77, 78, 79, 351, 352, 353, 354, 355, 359, 360, 361, 362, LCVDs that are not one of the sequences shown in 365, 366, 367, 368, 370, 371, 372, 373, 376, 377, 383, 386, 387, 388, 389, 392, 393, 394, 395, and 396, or (iii) LCVD of sequence number 5, and HCVDs that are not one of sequence numbers 15, 18, 19, 22, 24, 25, 32, 33, 34, 35, 36, 37, 38, 41, 43, 46, 47, 50, 318, 320, 322, 323, 324, 325, 328, 332, 333, 334, 336, 337, 343, 344, 345, 347, and 348, not included). (b) HCVD of one of the sequences shown in SEQ ID NOs. 80, 88-128, 397-406, or 397-406, and / or LCVD of one of the sequences shown in SEQ ID NOs. 81, 129-155, or 407-426 (the first ABD is (i) SEQ ID NOs. 80 and 81, (ii) SEQ ID NOs. 8 (iii) HCVDs that do not include HCVDs of number 0, and LCVDs that are not one of sequence numbers 131, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 147, 151, 152, 153, 411, 412, 417, 418, and 423, and (iii) LCVDs that do not include HCVDs of sequence number 81, and HCVDs that are not one of sequence numbers 91, 101, 103, 112, 113, 116, 119, 122, 123, 125, and 126, and (c) HCVD of one of the sequences shown in sequence number 156, sequence numbers 164-202, or sequence numbers 427-457, and / or LCVD of one of the sequences shown in sequence number 157, sequence numbers 203-233, or sequence numbers 458-492 (the first ABD is (i) the HCVD of sequence numbers 156 and 157, (ii) the HCVD of sequence number 156, and sequence numbers 204, 211, 213, 215, 218, 220, 221, 222, 223, 224, 225, 228, 229, 231, 232, 233, 459, 460, 462, 464, 465, 466 The ABPC according to claim 1, comprising one of the following: (iii) an LCVD that is not one of 467, 468, 469, 470, 473, 474, 485, 488, 489, and 491, or (iii) the LCVD of sequence number 157, and not an HCVD that is not one of the sequences shown in sequence numbers 164, 167, 168, 171, 172, 175, 178, 180, 181, 183, 196, 197, 200, 201, 427, 428, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445, 446, 447, 449, 451, 454, 456, and 457.

10. The ABPC according to any one of claims 1 to 9, wherein the ABPC is degraded in the target mammalian cells after internal translocation of the ABPC by the target mammalian cells, and / or the ABPC further comprises a conjugated toxin, radioisotope, drug, or small molecule, and / or the ABPC provides an increase in toxin release in the target mammalian cells compared to the same amount of control ABPC.

11. The ABPC according to claim 10, wherein the ABPC provides at least a 20%, 50%, 2x, or 5x increase in toxin release compared to the same amount of control ABPC, and / or the ABPC provides an increase in the killing of target mammalian cells.

12. The ABPC according to claim 11, wherein the composition results in at least a 20%, 50%, 2-fold, or 5-fold increase in killing the target mammalian cells compared to the same amount of control ABPC.

13. The ABPC according to any one of claims 1 to 12, wherein the ABPC provides an increase in endolysosome delivery of target mammalian cells compared to the same amount of control ABPC, and optionally the increase is at least 20%, 50%, 2-fold, or 5-fold.

14. The ABPC according to any one of claims 1 to 13, wherein the ABPC does not cause, or does not cause, a detectable decrease in the level of TROP2 presented on the surface of the target mammalian cells compared to the same amount of control ABPC.

15. The ABPC according to any one of claims 1 to 14, wherein the ABPC further comprises a second ABD.

16. The ABPC according to any one of claims 1 to 15, wherein the ABPC is conjugated with a drug.

17. A kit comprising at least one dose of the ABPC described in any one of claims 1 to 16.

18. A method for treating cancer characterized by having a population of cancer cells having a predetermined level of TROP2 or a TROP2 epitope presented on their surfaces, comprising administering a therapeutically effective amount of ABPC according to any one of claims 1 to 16 to a subject identified as having cancer characterized by having a population of cancer cells.

19. A method for reducing the volume of a tumor in a subject, wherein the tumor is characterized by having TROP2-positive cancer cells, and the method comprises administering a therapeutically effective amount of the ABPC described in any one of claims 1 to 16 to the subject identified as having cancer characterized by the cancer cells.

20. A method for inducing cell death in TROP2-positive cancer cells in a subject, comprising administering a therapeutically effective amount of ABPC according to any one of the claims, or any one of the claims 1 to 16, to a subject identified as having the cancer.

21. A method for reducing the risk of metastasis or additional metastasis in a subject having TROP2-positive cancer, comprising administering a therapeutically effective dose of ABPC according to any one of claims 1 to 16 to the subject identified as having the cancer.

22. (a) to (f): (a) Arrival / Departure Times: 18+59, 19+59, 32+59, 34+59, 35+59, 36+59, 37+59, 38+59, 18+62, 19+62, 38+62, 19+65, 18+66, 19+66, 35+71, 38+71, 34+73, 19+75, 35+75, 36+75, 37+75, 38+75, 19+366, 32+366, 35+366, 36+366, 18+376, 32+376, 34+376, 35+376, 36+376, 37+376, 38+376, 18+351 , 19+351, 32+351, 34+351, 35+351, 36+351, 37+351, 38+351, 18+352, 19+352, 32+352, 34+352, 35+352, 36+352, 37+352, 38+352, 32+353, 34+353, 35+353, 37+353, 38+353, 18+361, 19+361, 32+361, 34+361, 35+361, 36+361, 37+361, 38+361, 18+365, 32+365, 34+365, 35+36 5, 36+365, 37+365, 38+365, 18+366, 19+366, 32+366, 34+366, 35+366, 36+366, 37+366, 38+366, 18+388, 32+388, 34+388, 35+388, 37+388, 38+388, 18+389, 19+389, 32+389, 34+389, 35+389, 36+389, 37+389, 38+389, 18+394, 32+394, 34+394, 35+394, 36+394, 37+394, 38+3 94, 101+134, 119+134, 122+134, 119+142, 119+143, 122+143, 116+148, 116+151, 119+151, 122+151, 196+223, 197+223, 200+223, 196+224, 197+224, 200+224, 196+225, 197+225, 200+225, 196+226, 197+226, 196+229, 197+229, 200+229, 196+233, 197+233, 197+460, 197+ One of the pairs of HCVD and LCVD sequences shown in one of the pairs 462, 197+460, and 197+462, (b) HCVD sequences and LCVD sequences having the same HCVD and LCVD as the HCVD sequences and LCVD sequences listed in one of the pairs of sequence numbers listed in (a), and HCVD sequences and LCVD sequences that are at least 90%, at least 95%, or at least 98% identical. (c) HCVD sequences and LCVD sequences (determined by the Kabat system) having the same HCVD and LCVD as the HCVD sequence and LCVD sequence shown in one of the pairs of sequence numbers listed in (a), and HCVD sequences and LCVD sequences that are at least 90%, at least 95%, or at least 98% identical. (d) HCVD sequences and LCVD sequences (determined by the IMGT system) that have the same HCVD and LCVD as the HCVD sequence and LCVD sequence shown in one of the pairs of sequence numbers listed in (a), respectively, and HCVD sequences and LCVD sequences that are at least 90%, at least 95%, or at least 98% identical. (e) HCVD sequences and LCVD sequences having the same HCVD and LCVD as the HCVD sequences and LCVD sequences shown in one of the pairs of sequence numbers listed in (a) (determined by additive Kabat-IMGT method), and HCVD sequences and LCVD sequences that are at least 90%, at least 95%, or at least 98% identical, (f) A TROP2-specific binding protein construct comprising a heavy chain variable domain (HCVD) and a light chain variable domain (LCVD), each comprising one of the following: an HCVD sequence and an LCVD sequence having the same HCVD and LCVD as those shown in one of the pairs of sequence numbers listed in (a) (determined by additive Kabat-IMGT); and an HCVD sequence and an LCVD sequence that are at least 98% identical.

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