LRRC15-binding protein constructs and their uses

Abstract

Provided herein are antigen-binding protein constructs (ABPCs), antibodies, and antibody-drug conjugates (ADCs) that bind to LRRC15, and uses thereof. Provided herein are pharmaceutical compositions comprising an effective amount of ABPC, the ABPC comprising a first antigen-binding domain capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed 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.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 415,594, filed October 12, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named "LRRC15_PRO_SL.XML". The XML file was created on Sep. 22, 2023, and is 1,035,741 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the field of biotechnology, and more particularly to antigen-binding molecules. Summary of the Invention

[0004] Antibody-drug-conjugates (ADCs) have been designed to combat a variety of diseases. One particular advantage of this approach is the ability for ADCs to have cytostatic or cytotoxic effects. Despite years of development, improved ADCs are desirable.

[0005] The present invention is based on novel antigen binding protein constructs (ABPCs) and antibodies (Abs) that exhibit increased internalization into mammalian cells, increased endolysosomal delivery, increased toxin release inside such cells, increased efficacy against conditions including cancer, and increased target mammalian cell killing.

[0006] Provided herein is a pharmaceutical composition comprising an effective amount of an ABPC, the ABPC comprising a first antigen-binding domain capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD is faster at a pH of about 4.0 to about 6.5 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.

[0007] Non-limiting examples of LRRC15-specific ABPCs and antibodies include those containing the variable domains and / or CDRs disclosed in WO2022 / 216653A1 (Mythic Therapeutics, incorporated herein by reference), including those ABPCs, antibodies, variable domains, and / or CDRs present in MYT2737 and MYT3315.

[0008] Prompted by these initial findings and the reasoning that anti-LRRC15-ADCs may prove useful against both LRRC15+ tumors as well as LRRC15 tumors associated with LRRC15+ stromal cells, Applicants produced the novel anti-LRRC15 antibodies disclosed herein.

[0009] As further detailed herein, the following are pH-dependent, anti-LRRC15 antibodies that exhibit strong target-specific binding at physiological pH, enhanced dissociation at acidic pH, enhanced internalization into LRRC15+ cells, and increased cytotoxicity when complexed or conjugated with a toxic payload: Non-limiting examples of ABPCs are: MYT2737 (HCV = SEQ ID NO: 1, LCV = SEQ ID NO: 64), MYT3315 (HCV = SEQ ID NO: 84, LCV = SEQ ID NO: 154), MYT8391 (HCV = SEQ ID NO: 430, LCV = SEQ ID NO: 455), MYT8415 (HCV = SEQ ID NO: 382, ​​LCV = SEQ ID NO: 489), MYT8417 (HCV = SEQ ID NO: 382, ​​LCV = SEQ ID NO: 491), MYT8483 (HCV = SEQ ID NO: 516, LCV = SEQ ID NO: 522), MYT9776 (HCV = SEQ ID NO: 571, LCV = SEQ ID NO: 517), MYT8094 (HCV = SEQ ID NO: 576, LCV = SEQ ID NO: 581), MYT9521 (HCV = SEQ ID NO: 576, LCV = SEQ ID NO: 639), MYT9731 (HCV = SEQ ID NO: 576, LCV = SEQ ID NO: 700), MYT8416 (HCV = SEQ ID NO: 382, ​​LCV = SEQ ID NO: 490), MYT8500 (HCV = SEQ ID NO: 570, LCV = SEQ ID NO: 522), MYT9523 (HCV = SEQ ID NO: 576, LCV = SEQ ID NO: 641), MYT4174 (HCV = SEQ ID NO: 84, LCV = SEQ ID NO: 177), and MYT9507 (HCV = SEQ ID NO: 576, LCV = SEQ ID NO: 625).

[0010] In some embodiments, anti-LRRC15 The ABPC has the same heavy and light chains, heavy and light chain variable domains, and / or heavy and light chain CDRs (e.g., as determined by the Kabat, Chothia, and / or IMGT numbering schemes) as MYT2737-c (comprising SEQ ID NOs: 724 & 725); MYT3315-c (comprising SEQ ID NOs: 726 & 727); MYT8391-c (comprising SEQ ID NOs: 728 & 729); MYT8415-c (comprising SEQ ID NOs: 730 & 731); MYT8417-c (comprising SEQ ID NOs: 730 & 732); MYT8483-c (comprising SEQ ID NOs: 733 & 734); MYT9776-c (comprising SEQ ID NOs: 735 & 736); MYT9521-c (comprising SEQ ID NOs: 737 & 738); MYT9731-c (comprising SEQ ID NOs: 737 & 739); or MYT8094-c (comprising 737 & 740). Each of the foregoing is suitable for toxin conjugation at various positions, including position 205 of the respective light chain (i.e., the sequence set forth in one of SEQ ID NOs: 725, 727, 729, 731, 732, 734, 736, 738, 739, and 740). In some embodiments, position 205 of the light chain includes an amino acid other than cysteine ​​(e.g., valine, leucine, isoleucine, or alanine). In some embodiments, toxin conjugation may be made at other positions known to those of skill in the art. In still other embodiments, the toxin may be attached, for example, via a hinge bond or any other suitable attachment methodology.

[0011] In some embodiments, the first ABD (also "LRRC15-binding domain") comprises one of the heavy chain variable (HCV) domains: (a) the HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. The first ABD may also comprise the HCV domain of MYT2737 (HC = SEQ ID NO: 1, LC = SEQ ID NO: 64) or MYT3315 (HC = SEQ ID NO: 84, LC = SEQ ID NO: 154). In some embodiments, the first ABD may comprise the three HCDRs of MYT2737 or MYT3315. As used herein, "15G7," "24D9," and "29F1" each refer to a newly discovered rabbit monoclonal antibody (RabMAb) clone and its corresponding humanized counterpart. When preceded by "hu" (e.g., "hu15G7"), a humanized clone is intended.

[0012] In some embodiments, the anti-LRRC15 antibody comprises a heavy chain and a light chain having the polypeptide sequence set forth in SEQ ID NO: 724&725;726&727;728&729;730&731;730&732;733&734;735&736;737&738;737&739; or 737&740. In other embodiments, the anti-LRRC15 antibody comprises a heavy chain and a light chain having the polypeptide sequence set forth in SEQ ID NO: 724&725;726&727;728&729;730&731;730&732;733&734;735&736;737&738;737&739; or 737&740, wherein position 205 of each light chain is not a cysteine. In some embodiments, the anti-LRRC15 antibody comprises a light chain having a valine, leucine, or isoleucine at position 205.

[0013] In some embodiments, an anti-LRRC15 antibody may comprise heavy and light chains that include preferred "developable mutations," e.g., as described in Table 33. As used herein, "developable mutations" refers to changes in the amino acid sequence of an ABPC, including antibodies, that tend to improve certain biophysical properties (e.g., reduced likelihood of aggregation). In other embodiments, the heavy and light chains may include conservative amino acid substitutions for preferred developable mutations.

[0014] In some embodiments, a composition comprising an ABPC or antibody provides one or more of the following: an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of a control antibody; an increase in target mammalian cell killing compared to a composition comprising the same amount of a control antibody; and / or an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of a control antibody.

[0015] In some embodiments, a composition comprising the antibody results in a reduced or undetectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells compared to a composition comprising the same amount of a control antibody.

[0016] In some embodiments, the antibody is degraded in the target mammalian cell after internalization of the antibody by the target mammalian cell. In some embodiments, the target mammalian cell is a cancer cell. In some embodiments, the antibody is cytotoxic or cytostatic to the target mammalian cell. In some embodiments, the antibody has an avidity that results in high selectivity for cancer cells over non-cancerous cells.

[0017] In some embodiments, the antibody is cross-reactive with one or both of non-human primate LRRC15 and human LRRC15; or rat LRRC15 and mouse LRRC15.

[0018] In some embodiments, the half-life of the antibody in vivo is increased compared to the half-life of a control antibody in vivo.

[0019] In some embodiments, the first ABD comprises one of the following light chain variable (LCV) domains: (a) the LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. The first ABD may also comprise the LCV domain of MYT2737 or MYT3315.

[0020] In some embodiments, the first ABD comprises (a) the HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; and / or the LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. If the wild-type amino acid is already histidine, alanine may be substituted instead.

[0021] In some embodiments, the HCV domain comprises one of: (a) the HCV domain of 15G7 comprising SEQ ID NO: 378, or a humanized version thereof (e.g., SEQ ID NO: 382); (b) the HCV domain of 24D9 comprising SEQ ID NO: 512, or a humanized version thereof (e.g., SEQ ID NO: 516); and (c) the HCV domain of 29F1 comprising SEQ ID NO: 572, or a humanized version thereof (e.g., SEQ ID NO: 576). In some embodiments, the LCV domain comprises one of: (a) the LCV domain of 15G7 comprising SEQ ID NO: 451, or a humanized version thereof (e.g., SEQ ID NO: 455); (b) the LCV domain of 24D9 comprising SEQ ID NO: 518, or a humanized version thereof (e.g., SEQ ID NO: 522); and (c) the LCV domain of 29F1 comprising SEQ ID NO: 577, or a humanized version thereof (e.g., SEQ ID NO: 581).

[0022] In some embodiments, the first LRRC15-binding domain comprises one of the following HCV domains: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573-575, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0023] In some embodiments, the first LRRC15 binding domain comprises one of the following LCV domains: (a) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452 to 454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452 to 454 collectively substituted with histidine; (b) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 519 to 521 collectively substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 578 to 580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0024] In some embodiments, the first LRRC15-binding domain comprises: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; and / or an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452-454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452-454 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and / or and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 519 to 521 optionally substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573 to 575, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 573 to 575 optionally substituted with histidine, aspartic acid, or glutamic acid; and / or an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 578 to 580 optionally substituted with histidine, aspartic acid, or glutamic acid.

[0025] In some embodiments, the first ABD is selected from the group consisting of: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises a histidine (or an alanine if a histidine is already present) at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 31, 56, 59, and 99 in SEQ ID NO:572 or SEQ ID NO:576; and (d) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises a histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of 31, 56, 59, and 99 in SEQ ID NO:572 or SEQ ID NO:576. In some embodiments, the first ABD comprises: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; and (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises a histidine, aspartic acid (D), or glutamic acid (E) at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0026] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises histidines at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair of positions selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; and (b) an HCV domain that comprises a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises histidines at one or more positions in SEQ ID NO: 512 or SEQ ID NO: 516. In some embodiments, the first ABD comprises one of: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises histidines at two or more positions in SEQ ID NO: 451 or SEQ ID NO: 455, including one pair of positions selected from the group consisting of: 30, 32; 30, 92; 30, 93; 30, 96; 32, 92; 32, 93; 32, 96; 92, 93; 92, 96 and 93, 96; and (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCV domain comprises histidines at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522.

[0027] In some embodiments, the first ABD comprises (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO: 512 or SEQ ID NO: 516; and / or or (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCV domain comprises a histidine at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522 selected from the group consisting of 35 and 97; and (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCV domain comprises a D position selected from 31, 56, and 99; or an E position 59 in SEQ ID NO: 572 or SEQ ID NO: 576. and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56 in SEQ ID NO: 577 or SEQ ID NO: 581; and E positions selected from 51 and 56.

[0028] In some embodiments, the first ABD comprises (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising the sequence of SEQ ID NO: 512, one of SEQ ID NOs: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, one of SEQ ID NOs: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695; and / or the first ABD comprises (a) an LCV domain having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456 to 485, and one of SEQ ID NOs: 486 to 511; (b) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with an L35H substitution), or SEQ ID NO: 518 (with a G97H substitution); and (c) one of the LCV domains comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625 to 652, and one of SEQ ID NOs: 696 to 723.

[0029] In some embodiments, the first ABD comprises one of (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, and the first ABD comprises (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382, ​​and a LCV domain having the sequence of SEQ ID NO: 461, SEQ ID NO: 462, or (ii) an LCV domain comprising the sequence of SEQ ID NO: 451 or SEQ ID NO: 455, and a LCV domain comprising the sequence of SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 396, one of SEQ ID NO: 403, one of SEQ ID NOs: 417 to 419, one of SEQ ID NOs: 423 to 424, one of SEQ ID NOs: 426 to 430, one of SEQ ID NOs: 438 to 440, SEQ ID NO: 444, and SEQ ID NO: 450. (b) an HCV domain comprising the sequence of SEQ ID NO: 512, one of SEQ ID NOs: 516, one of SEQ ID NOs: 523 to 567, and one of SEQ ID NOs: 568 to 571; and / or an HCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with a L35H substitution), or SEQ ID NO: 518 (with a G97H substitution), wherein the first ABD does not comprise (i) an HCV domain comprising the sequence of SEQ ID NO: 512 or SEQ ID NO: 516, and an HCV domain comprising the sequence of SEQ ID NO: 518 (with a L35H substitution). or (ii) an LCV domain comprising a sequence other than one of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H), and SEQ ID NO: 518 (with G97H); or (ii) an HCV domain comprising a sequence other than one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NO: 564;and (c) does not include an HCV domain comprising the sequence of SEQ ID NO: 572 or SEQ ID NO: 576, and / or an LCV domain comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625 to 652, and one of SEQ ID NOs: 696 to 723, wherein the first ABD does not include (i) an HCV domain comprising the sequence of SEQ ID NO: 572 or SEQ ID NO: 576, and an LCV domain comprising a sequence other than one of SEQ ID NOs: 628, 629, 632, 638, and 642; or (ii) an LCV domain comprising the sequence of SEQ ID NO: 577 or SEQ ID NO: 581, and an HCV domain comprising a sequence other than one of SEQ ID NOs: 587, 598, 611, and 672;

[0030] In some embodiments, the ABPC is degraded in the target mammalian cell after internalization of the ABPC by the target mammalian cell, hi some embodiments, the ABPC comprises a conjugated toxin, radioisotope, drug, or small molecule.

[0031] In some embodiments, the composition provides an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 20% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0032] In some embodiments, the composition provides an increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 20% increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC.

[0033] In some embodiments, the composition provides an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 20% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0034] In some embodiments, the composition causes a smaller reduction in the level of LRRC15 displayed on the surface of target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition does not cause a detectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells.

[0035] Also provided herein is a pharmaceutical composition comprising an effective amount of an antigen binding protein construct (ABPC), the first ABD being capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed 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 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, or the dissociation constant (K) 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. D ) at a pH of about 7.0 to about 8.0 D and (b) the composition provides one or more of: an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC; an increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC; and an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0036] In some embodiments, the first ABD comprises (a) an HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises (a) an LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0037] In some embodiments, the first ABD comprises one of: (a) the HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; and / or the LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0038] In some embodiments, the HCV domain includes one of: (a) the HCV domain of 15G7, or a humanized version thereof, comprising SEQ ID NO: 378 (e.g., SEQ ID NO: 382); (b) the HCV domain of 24D9, or a humanized version thereof, comprising SEQ ID NO: 512 (e.g., SEQ ID NO: 516); and (c) the HCV domain of 29F1, or a humanized version thereof, comprising SEQ ID NO: 572 (e.g., SEQ ID NO: 576); in some embodiments, the LCV domain includes one of: (a) the LCV domain of 15G7, or a humanized version thereof, comprising SEQ ID NO: 451 (e.g., SEQ ID NO: 455); (b) the LCV domain of 24D9, or a humanized version thereof, comprising SEQ ID NO: 518 (e.g., SEQ ID NO: 522); and (c) the LCV domain of 29F1, or a humanized version thereof, comprising SEQ ID NO: 577 (e.g., SEQ ID NO: 581).

[0039] In some embodiments, the first ABD comprises one of: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573-575, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0040] In some embodiments, the first LRRC15 binding domain comprises one of the following LCV domains: (a) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452 to 454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452 to 454 collectively substituted with histidine; (b) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 519 to 521 collectively substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 578 to 580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0041] In some embodiments, the first LRRC15-binding domain comprises: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; and / or an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452-454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452-454 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and / or and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 519 to 521 optionally substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573 to 575, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 573 to 575 optionally substituted with histidine, aspartic acid, or glutamic acid; and / or an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 578 to 580 optionally substituted with histidine, aspartic acid, or glutamic acid.

[0042] In some embodiments, the first ABD comprises: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCV domain comprises a histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of 31, 56, 59, and 99 in SEQ ID NO: 572 or SEQ ID NO: 576; and (d) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCV domain comprises a histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO: 516.

[0043] In some embodiments, the first ABD is selected from the group consisting of: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO: 451 or SEQ ID NO: 455; (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCV domain comprises , and histidine at one or more positions selected from the group consisting of 35 and 97 in SEQ ID NO: 518 or SEQ ID NO: 522; and (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of 27, 28, 31, 51, 52, and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0044] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises histidines at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; and (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises histidines at one or more positions in SEQ ID NO: 512 or SEQ ID NO: 516. In some embodiments, the first ABD comprises one of: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises histidines at two or more positions in SEQ ID NO: 451 or SEQ ID NO: 455, including one pair of positions selected from the group consisting of: 30, 32; 30, 92; 30, 93; 30, 96; 32, 92; 32, 93; 32, 96; 92, 93; 92, 96; 93, 96; and (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCV domain comprises histidines at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522.

[0045] In some embodiments, the first ABD comprises: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises one or more positions selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO:512 or SEQ ID NO:516. contains histidines at multiple positions; and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain contains histidines at one or more positions in SEQ ID NO:518 or SEQ ID NO:522 selected from the group consisting of 35 and 97; and (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain contains histidines at one or more positions selected from the group consisting of 35 and 97 in SEQ ID NO:518 or SEQ ID NO:522. and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises a histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of 27, 28, 31, 51, 52, and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0046] In some embodiments, the first ABD comprises one of the following HCV domains: (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising the sequence of SEQ ID NO: 512, one of SEQ ID NOs: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, one of SEQ ID NOs: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695.

[0047] In some embodiments, the first ABD comprises (a) an LCV domain having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511; (b) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H); and (c) one of the LCV domains comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625-652, and one of SEQ ID NOs: 696-723.

[0048] In some embodiments, the first ABD comprises one of (a) an HCV domain comprising the sequence of SEQ ID NO: 378, any one of SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain of SEQ ID NO: 451, any one of SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and any one of SEQ ID NOs: 486-511, and the first ABD comprises (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382, ​​and a LCV domain of SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO 64, SEQ ID NO: 466, one of SEQ ID NO: 467, one of SEQ ID NOs: 477 to 478, and one of SEQ ID NOs: 480 to 482; and (ii) an LCV domain comprising the sequence of SEQ ID NO: 451 or SEQ ID NO: 455, and an HCV domain comprising a sequence that does not include one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NOs: 564; (b) SEQ ID NOs: 512, 516, 523 to 56 and (ii) an HCV domain comprising the sequence of any one of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with an L35H substitution), or SEQ ID NO: 518 (with a G97H substitution), wherein the first ABD is selected from the group consisting of: (i) an HCV domain comprising the sequence of SEQ ID NO: 512 or SEQ ID NO: 516, and an LCV domain comprising a sequence other than one of SEQ ID NO: 518 (with an L35H substitution) and SEQ ID NO: 518 (with a G97H substitution); and (ii) an HCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with an L35H substitution), or SEQ ID NO: 518 (with a G97H substitution); (c) an LCV domain comprising the sequence of SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H), and an HCV domain comprising a sequence that does not include one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NO: 564; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, any one of SEQ ID NOs: 576, one of SEQ ID NOs: 582 to 624, and one of SEQ ID NOs: 653 to 695;and / or does not include any one of the LCV domains of SEQ ID NOs: 577, 581, 625 to 652, or 696 to 723, and the first ABD does not include (i) an HCV domain comprising the sequence of SEQ ID NO: 572 or SEQ ID NO: 576, and an LCV domain comprising a sequence other than one of SEQ ID NOs: 628, 629, 632, 638, and 642; and (ii) an LCV domain comprising the sequence of SEQ ID NO: 577 or SEQ ID NO: 581; and an HCV domain comprising a sequence other than one of SEQ ID NOs: 587, 598, 611, and 672;

[0049] In some embodiments, the composition provides an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 20% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0050] In some embodiments, the composition provides an increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 20% increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC. In some embodiments, the composition provides an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0051] In some embodiments, the composition provides at least a 20% increase in endolysosomal delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides at least a 50% increase in endolysosomal delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides at least a 2-fold increase in endolysosomal delivery in target mammalian cells compared to a composition containing the same amount of control ABPC. In some embodiments, the composition provides at least a 5-fold increase in endolysosomal delivery in target mammalian cells compared to a composition containing the same amount of control ABPC.

[0052] In some embodiments, the composition causes a smaller reduction in the level of LRRC15 displayed on the surface of target mammalian cells compared to a composition comprising the same amount of control ABPC.In some embodiments, the composition causes no detectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells.In some embodiments, the target mammalian cells are cancer cells.

[0053] In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10% faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 3 times faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10 times faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the K D is the K of ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the K of the ABD at a pH of about 4.0 to about 6.5 is at least 10% greater than D is the K of ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the K of the ABD at a pH of about 4.0 to about 6.5 is at least three times greater than D is the K of ABD at a pH of about 7.0 to about 8.0 D is at least 10 times larger than

[0054] In some embodiments, ABPC is cytotoxic or cytostatic to target mammalian cells. In some embodiments, ABPC is cross-reactive with non-human primate LRRC15 and human LRRC15. In some embodiments, ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, and one or both of rat LRRC15 and mouse LRRC15. In some embodiments, ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15. In some embodiments, the ABD binds to an epitope of LRRC15 present on the surface of cells from Old World monkeys.

[0055] In some embodiments, the ABPC comprises a single polypeptide. In some embodiments, the ABD is selected from the group consisting of a VH domain, a VHH domain, a VNAR domain, and an scFv. In some embodiments, the ABPC is BiTe, (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scdiabody, a scdiabody-CH3, a scFv-CH-CL-scFv, an HSAbody, a scdiabody-HSA, or a tandem-scFv.

[0056] In some embodiments, an ABPC comprises two or more polypeptides, such as an antibody, VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knobs-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, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, dock and lock lock), ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAB, and PROTAC.

[0057] In some embodiments, the ABPC or ABD comprises a proteolysis-targeting antibody (PROTAB) (e.g., as described in Marei et al., "Antibody targeting of E3 ubiquitin ligases for receptor degradation." Nature, October 6, 2022). In some embodiments, the PROTAB or PROTAC can tether a cell surface E3 ubiquitin ligase to a transmembrane protein, resulting in targeted degradation both in vitro and in vivo.

[0058] In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a cleavable linker, hi some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a non-cleavable linker.

[0059] In some embodiments, the half-life of ABPC in vivo is increased compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 5% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 10% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 30% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 50% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 70% to about 95% compared to the half-life of a control ABPC in vivo. In other embodiments, the in vivo half-life may be decreased.

[0060] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 three-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than three-fold faster. D ) is the K at a pH of about 7.0 to about 8.0 D is less than three times larger than

[0061] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 two-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is no more than two-fold faster. D ) is the K at a pH of about 7.0 to about 8.0 D is less than twice as large as

[0062] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 ) is the K at a pH of about 7.0 to about 8.0 D is less than one time larger than

[0063] In some embodiments, the control ABPC is selected from one of: (a) 15G7; (b) 24D9; and (c) 29F1. In some embodiments, the control ABPC is selected from one of: (a) hu15G7; (b) hu24D9; and (c) hu29F1.

[0064] In some embodiments, the ABPC comprises a second ABD.

[0065] Also provided herein are kits containing at least one dose of any of the pharmaceutical compositions described herein.

[0066] Also provided herein is an antigen binding protein construct (ABPC) comprising a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed 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; or (b) the dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5 is D ) at a pH of about 7.0 to about 8.0 D is greater than.

[0067] In some embodiments, the first ABD comprises (a) an HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises (a) an LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0068] In some embodiments, the first ABD comprises one of: (a) the HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; and / or the LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0069] In some embodiments, the HCV domain comprises one of: (a) the HCV domain of 15G7 comprising SEQ ID NO: 378, or a humanized version thereof (e.g., SEQ ID NO: 382); (b) the HCV domain of 24D9 comprising SEQ ID NO: 512, or a humanized version thereof (e.g., SEQ ID NO: 516); and (c) the HCV domain of 29F1 comprising SEQ ID NO: 572, or a humanized version thereof (e.g., SEQ ID NO: 576). In some embodiments, the LCV domain comprises one of: (a) the LCV domain of 15G7 comprising SEQ ID NO: 451, or a humanized version thereof (e.g., SEQ ID NO: 455); (b) the LCV domain of 24D9 comprising SEQ ID NO: 518, or a humanized version thereof (e.g., SEQ ID NO: 522); and (c) the LCV domain of 29F1 comprising SEQ ID NO: 577, or a humanized version thereof (e.g., SEQ ID NO: 581).

[0070] In some embodiments, the first ABD comprises one of the following HCV domains: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573-575, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first LRRC15 binding domain comprises one of the following LCV domains: (a) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452 to 454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452 to 454 collectively substituted with histidine; (b) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 519 to 521 collectively substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 578 to 580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0071] In some embodiments, the first LRRC15-binding domain comprises: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; and / or an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452-454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452-454 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and / or and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 519 to 521 optionally substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573 to 575, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 573 to 575 optionally substituted with histidine, aspartic acid, or glutamic acid; and / or an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 578 to 580 optionally substituted with histidine, aspartic acid, or glutamic acid.

[0072] In some embodiments, the first ABD comprises: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 30, 31, 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; and (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of D positions selected from 31, 56, and 99; and E positions selected from 59 in SEQ ID NO:572 or SEQ ID NO:576. In some embodiments, the first ABD comprises: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; and (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises one of the following: a histidine at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0073] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises histidines at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; and (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises histidines at one or more positions in SEQ ID NO: 512 or SEQ ID NO: 516.

[0074] In some embodiments, the first ABD comprises (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO:512 or SEQ ID NO:516; and / or (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises a histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522 selected from the group consisting of 35 and 97; and (d) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises a histidine at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of D positions 31, 56 and 99; and E positions selected from 59. and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0075] In some embodiments, the first ABD comprises (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising the sequence of SEQ ID NO: 512, one of SEQ ID NOs: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) one of HCV domains comprising the sequence of SEQ ID NO: 572, one of SEQ ID NOs: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695; and / or the first ABD comprises (a) an LCV domain having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456 to 485, and one of SEQ ID NOs: 486 to 511; (b) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H); and (c) one of the LCV domains comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625 to 652, and one of SEQ ID NOs: 696 to 723.

[0076] In some embodiments, the first ABD comprises (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or one of LCV domains having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, and the first ABD comprises (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382, ​​and one of SEQ ID NOs: 461, 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503 (ii) an LCV domain comprising a sequence other than SEQ ID NO: 64, SEQ ID NO: 466, one of SEQ ID NOs: 467, one of SEQ ID NOs: 477 to 478, and one of SEQ ID NOs: 480 to 482; or (ii) an LCV domain comprising a sequence other than SEQ ID NO: 451 or SEQ ID NO: 455 and one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NO: 564; (b) an HCV domain comprising a sequence other than SEQ ID NO: 512, SEQ ID NO: 516 and / or an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H), and the first ABD does not include (i) an HCV domain comprising the sequence of SEQ ID NO: 512 or SEQ ID NO: 516, and an LCV domain comprising a sequence other than one of SEQ ID NO: 518 (with an L35H substitution) and SEQ ID NO: 518 (with a G97H substitution). V domain; or (ii) an HCV domain comprising the sequence of SEQ ID NO: 518 or SEQ ID NO: 518 or SEQ ID NO: 522, and a sequence that does not include one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NO: 564; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, or one of SEQ ID NOs: 582 to 624, or one of SEQ ID NOs: 653 to 695;and / or does not contain an LCV domain comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625 to 652, and one of SEQ ID NOs: 696 to 723, and the first ABD does not contain (i) an HCV domain comprising the sequence of SEQ ID NO: 572 or SEQ ID NO: 576, and an LCV domain comprising a sequence other than one of SEQ ID NOs: 628, 629, 632, 638, and 642; or (ii) an LCV domain comprising the sequence of SEQ ID NO: 577 or SEQ ID NO: 581, and an HCV domain comprising a sequence other than one of SEQ ID NOs: 587, 598, 611, and 672;

[0077] In some embodiments, the ABPC is degraded in the target mammalian cell after internalization of the ABPC by the target mammalian cell, hi some embodiments, the ABPC comprises a conjugated toxin, radioisotope, drug, or small molecule.

[0078] In some embodiments, a composition comprising ABPC provides an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 20% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 50% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 2-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 5-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0079] In some embodiments, compositions comprising ABPC provide an increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 20% increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 50% increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 2-fold increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 5-fold increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC.

[0080] In some embodiments, a composition comprising ABPC provides an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, ABPC provides at least a 20% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 50% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 2-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 5-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0081] In some embodiments, a composition comprising ABPC causes a smaller reduction in the level of LRRC15 displayed on the surface of target mammalian cells compared to a composition comprising the same amount of control ABPC.In some embodiments, a composition comprising ABPC does not cause a detectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells.

[0082] Also provided herein is an antigen binding protein construct (ABPC), comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed 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 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, or the dissociation constant (K) 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. D ) at a pH of about 7.0 to about 8.0 D and (b) the composition provides one or more of: an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC; an increase in target mammalian cell killing compared to a composition comprising the same amount of control ABPC; and an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0083] In some embodiments, the first ABD comprises (a) an HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises (a) an LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) an LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) an LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0084] In some embodiments, the first ABD comprises one of: (a) the HCV domain of 15G7, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 15G7, optionally with one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and / or the LCV domain of 24D9, optionally with one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; and / or the LCV domain of 29F1, optionally with one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0085] In some embodiments, the HCV domain comprises one of: (a) the HCV domain of 15G7 comprising SEQ ID NO: 378, or a humanized version thereof (e.g., SEQ ID NO: 382); (b) the HCV domain of 24D9 comprising SEQ ID NO: 512, or a humanized version thereof (e.g., SEQ ID NO: 516); and (c) the HCV domain of 29F1 comprising SEQ ID NO: 572, or a humanized version thereof (e.g., SEQ ID NO: 576). In some embodiments, the LCV domain comprises one of: (a) the LCV domain of 15G7 comprising SEQ ID NO: 451, or a humanized version thereof (e.g., SEQ ID NO: 455); (b) the LCV domain of 24D9 comprising SEQ ID NO: 518, or a humanized version thereof (e.g., SEQ ID NO: 522); and (c) the LCV domain of 29F1 comprising SEQ ID NO: 577, or a humanized version thereof (e.g., SEQ ID NO: 581).

[0086] In some embodiments, the first ABD comprises one of the following HCV domains: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573-575, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first LRRC15 binding domain comprises one of the following LCV domains: (a) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452 to 454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452 to 454 collectively substituted with histidine; (b) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 519 to 521 collectively substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 578 to 580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0087] In some embodiments, the first LRRC15-binding domain comprises: (a) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379-381, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine; and / or an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452-454, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 452-454 collectively substituted with histidine; (b) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513-515, respectively, optionally with a total of one or more amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and / or and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519 to 521, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 519 to 521 optionally substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 573 to 575, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 573 to 575 optionally substituted with histidine, aspartic acid, or glutamic acid; and / or an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 578 to 580, respectively, collectively with one or more amino acid positions in SEQ ID NOs: 578 to 580 optionally substituted with histidine, aspartic acid, or glutamic acid.

[0088] In some embodiments, the first ABD comprises: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 30, 31, 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; and (c) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of D positions selected from 31, 56, and 99; and E positions selected from 59 in SEQ ID NO:572 or SEQ ID NO:576. In some embodiments, the first ABD comprises (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455; and (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises one of the following: a histidine at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56 in SEQ ID NO: 577 or SEQ ID NO: 581; and an E position selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0089] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises histidines at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; and (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises histidines at one or more positions in SEQ ID NO: 512 or SEQ ID NO: 516.

[0090] In some embodiments, the first ABD comprises one of: (a) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises histidines at two or more positions in SEQ ID NO: 451 or SEQ ID NO: 455, including one pair of positions selected from the group consisting of: 30, 32; 30, 92; 30, 93; 30, 96; 32, 92; 32, 93; 32, 96; 92, 93; 92, 96 and 93, 96; and (b) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCV domain comprises histidines at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522.

[0091] In some embodiments, the first ABD comprises (a) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at one or more positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or SEQ ID NO: 382; (b) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises a histidine at one or more positions selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO:512 or SEQ ID NO:516; and / or (c) an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises a histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522 selected from the group consisting of 35 and 97; and (d) an HCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises a histidine at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of D positions 31, 56 and 99; and E positions selected from 59. and / or an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCV domain comprises histidine, aspartic acid, or glutamic acid at one or more positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0092] In some embodiments, the first ABD comprises one of the following HCV domains: (a) an HCV domain comprising the sequence of one of SEQ ID NOs: 378, 383-424, and 425-450; (b) an HCV domain comprising the sequence of SEQ ID NO: 512, 516, 523-567, and 568-571; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, 576, 582-624, and 653-695. In some embodiments, the first ABD comprises (a) an LCV domain having the sequence of SEQ ID NO: 451, one of SEQ ID NOs: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511; (b) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H); and (c) one of the LCV domains comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625-652, and one of SEQ ID NOs: 696-723.

[0093] In some embodiments, the first ABD comprises one of (a) an HCV domain comprising the sequence of SEQ ID NO: 378, one of SEQ ID NOs: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain having the sequence of SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, and the first ABD comprises (i) an HCV domain of SEQ ID NO: 378 or 382, ​​and a LCV domain having the sequence of SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: (ii) an LCV domain comprising a sequence other than one of SEQ ID NOs: 464, 466, 467, one of SEQ ID NOs: 477 to 478, and one of SEQ ID NOs: 480 to 482; or (ii) an LCV domain comprising a sequence other than one of SEQ ID NOs: 451 and one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NOs: 564; (b) an HCV domain comprising a sequence other than one of SEQ ID NOs: 512, 51 and / or an HCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID NO: 518 (with L35H), or SEQ ID NO: 518 (with G97H), and the first ABD does not comprise (i) an HCV domain comprising the sequence of SEQ ID NO: 512 or SEQ ID NO: 516, and one of SEQ ID NO: 518 (with L35H substitution) and SEQ ID NO: 518 (with G97H substitution). or (ii) an LCV domain comprising the sequence of SEQ ID NO: 518 or SEQ ID NO: 518 or SEQ ID NO: 522 and an HCV domain comprising a sequence that does not include one of SEQ ID NOs: 527 to 529, one of SEQ ID NOs: 535 to 536, one of SEQ ID NOs: 541 to 543, SEQ ID NO: 552, one of SEQ ID NOs: 559 to 560, and one of SEQ ID NO: 564; and (c) an HCV domain comprising the sequence of SEQ ID NO: 572, 576, or 582 to 624, or 653 to 695;and / or does not contain an LCV domain comprising the sequence of SEQ ID NO: 577, one of SEQ ID NOs: 581, one of SEQ ID NOs: 625 to 652, and one of SEQ ID NOs: 696 to 723, and the first ABD does not contain (i) an HCV domain comprising the sequence of SEQ ID NO: 572 or SEQ ID NO: 576, and an LCV domain comprising a sequence other than one of SEQ ID NOs: 628, 629, 632, 638, and 642; or (ii) an LCV domain comprising the sequence of SEQ ID NO: 577 or SEQ ID NO: 581, and an HCV domain comprising a sequence other than one of SEQ ID NOs: 587, 598, 611, and 672;

[0094] In some embodiments, a composition comprising ABPC provides an increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 20% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 50% increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 2-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 5-fold increase in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0095] In some embodiments, compositions comprising ABPC provide an increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 20% increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 50% increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 2-fold increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC. In some embodiments, compositions comprising ABPC provide at least a 5-fold increase in target mammalian cell killing compared to compositions comprising the same amount of control ABPC.

[0096] In some embodiments, a composition comprising ABPC provides an increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 20% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 50% increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 2-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC. In some embodiments, a composition comprising ABPC provides at least a 5-fold increase in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0097] In some embodiments, the composition comprising ABPC causes a smaller reduction in the level of LRRC15 displayed on the surface of target mammalian cells compared to the composition comprising the same amount of control ABPC.In some embodiments, the composition comprising ABPC causes no detectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells.In some embodiments, the target mammalian cells are cancer cells.

[0098] In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10% faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 3 times faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10 times faster than the dissociation rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the K D is the K of ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the K of the ABD at a pH of about 4.0 to about 6.5 is at least 10% greater than D is the K of ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the K of the ABD at a pH of about 4.0 to about 6.5 is at least three times greater than D is the K of ABD at a pH of about 7.0 to about 8.0 D is at least 10 times larger than

[0099] In some embodiments, ABPC is cytotoxic or cytostatic to target mammalian cells.

[0100] In some embodiments, the ABPC is cross-reactive with non-human primate LRRC15 and human LRRC15. In some embodiments, the ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, and one or both of rat LRRC15 and mouse LRRC15. In some embodiments, the ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15. In some embodiments, the ABD binds to an epitope of LRRC15 present on the surface of cells from Old World monkeys.

[0101] In some embodiments, the ABPC comprises a single polypeptide. In some embodiments, the ABD is selected from the group consisting of a VH domain, a VHH domain, a VNAR domain, and an scFv. In some embodiments, the ABPC is BiTe, (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scdiabody, a scdiabody-CH3, a scFv-CH-CL-scFv, an HSAbody, a scdiabody-HSA, or a tandem-scFv.

[0102] In some embodiments, an ABPC comprises two or more polypeptides, such as an antibody, VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knobs-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, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, dock and lock lock), ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAB, and PROTAC.

[0103] In some embodiments, the ABPC or ABD comprises a proteolysis-targeting antibody (PROTAB) (e.g., as described in Marei et al., "Antibody targeting of E3 ubiquitin ligases for receptor degradation." Nature, October 6, 2022). In some embodiments, the PROTAB or PROTAC can tether a cell surface E3 ubiquitin ligase to a transmembrane protein, resulting in targeted degradation both in vitro and in vivo.

[0104] In some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a cleavable linker, hi some embodiments, at least one polypeptide of ABPC is conjugated to a toxin, radioisotope, drug, or small molecule via a non-cleavable linker.

[0105] In some embodiments, the half-life of ABPC in vivo is increased compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 5% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 10% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 30% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 50% to about 95% compared to the half-life of a control ABPC in vivo. In some embodiments, the half-life of ABPC in vivo is increased by about 70% to about 95% compared to the half-life of a control ABPC in vivo.

[0106] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 three-fold faster than the dissociation rate at a pH of about 7.0 to about 8.0; and (c) the dissociation constant (K D ) is the K at a pH of about 7.0 to about 8.0 D It is less than three times larger than

[0107] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 two-fold faster than the dissociation rate at a pH of about 7.0 to 8.0; and (c) the dissociation constant (K D ) is the K at a pH of about 7.0 to about 8.0 D It is less than twice as large.

[0108] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (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 ) is the K at a pH of about 7.0 to about 8.0 D It is less than one time larger than

[0109] In some embodiments, the control ABPC is selected from one of: (a) 15G7 (comprising SEQ ID NOs: 378 and 451); (b) 24D9 (comprising SEQ ID NOs: 512 and 518); (c) 29F1 (comprising SEQ ID NOs: 572 and 577); (d) a parent antibody of MYT2737; and (e) a parent antibody of MYT3315.

[0110] In some embodiments, the ABPC comprises a second ABD.

[0111] Also provided herein are kits that include at least one dose of any of the ABPCs described herein.

[0112] Also provided herein are methods of treating a cancer characterized by having a population of cancer cells, cancer-associated fibroblasts (CAFs), and / or stromal cells that have LRRC15 or epitopes of LRRC15 displayed on their surface, 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 a cancer characterized by having a population of cancer cells, CAFs, and / or stromal cells.

[0113] Also provided herein is a method for reducing tumor volume in a subject, wherein the tumor is characterized by having a population of cancer cells, CAFs, and / or stromal cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, 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 a cancer characterized by having a population of cancer cells, CAFs, and / or stromal cells.

[0114] Also provided herein are methods of inducing cell death in cancer cells, CAFs, and / or stromal cells in a subject, wherein the cancer cells, CAFs, and / or stromal cells have LRRC15 or an epitope of LRRC15 displayed on their surface, 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 a cancer characterized by having a population of cancer cells, CAFs, and / or stromal cells.

[0115] In some embodiments, the cancer is a primary tumor or a metastasis. In some embodiments, the cancer is a non-T cell infiltrating tumor. In some embodiments, the cancer is a T cell infiltrating tumor.

[0116] Also provided herein are methods for reducing the risk of developing metastases or reducing the risk of developing additional metastases in a subject with cancer, wherein the cancer is characterized by having a population of cancer cells, CAFs, and / or stromal cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, the method comprising administering a therapeutically effective amount of any one of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having a cancer characterized by having a population of cancer cells, CAFs, and / or stromal cells. In some embodiments, the cancer is a sarcoma, such as a leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0117] As used herein, the term "antigen binding protein construct" refers to (i) a single polypeptide comprising at least one ABD, or (ii) a complex of two or more polypeptides (e.g., the same or different polypeptides) that together form at least one ABD. Non-limiting examples and embodiments of antigen binding protein constructs are described herein. Additional examples and embodiments of antigen binding protein constructs are known in the art.

[0118] A "multispecific antigen-binding protein construct" is an antigen-binding protein construct comprising two or more different ABDs that collectively specifically bind two or more different epitopes. The two or more different epitopes can 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 surface of different cells). In some aspects, the antigens are present on the surface of a cell. In some aspects, the multispecific antigen-binding protein construct binds to two different epitopes (i.e., a "bispecific antigen-binding protein construct"). In some aspects, the multispecific antigen-binding protein construct binds to three different epitopes (i.e., a "trispecific antigen-binding protein construct"). In some aspects, the multispecific antigen-binding protein construct binds to four different epitopes (i.e., a "tetraspecific antigen-binding protein construct"). In some aspects, the multispecific antigen-binding protein construct binds to five different epitopes (i.e., a "pentaspecific antigen-binding protein construct"). Each binding specificity may be present in any suitable valency. Non-limiting examples of multispecific antigen-binding protein constructs are described herein.

[0119] An "ABD" is one or more protein domains (e.g., formed from amino acids from a single polypeptide, or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that are capable of specifically binding to one or more different antigens). In some examples, an ABD can bind to an antigen or epitope with specificity and affinity similar to that of a natural antibody. In some embodiments, an ABD can be an antibody or fragment thereof. In some embodiments, an ABD can comprise an alternative scaffold. Non-limiting examples of ABDs are described herein. Additional examples of ABDs are known in the art. In some examples, an ABD can bind to a single antigen.

[0120] The term "antibody" is used herein in its broadest sense and includes specific types of immunoglobulin molecules that contain one or more ABDs that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins, such as 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. Additional examples of antibodies are known in the art.

[0121] The phrase "endosomal / lysosomal pathway" refers to the network of endosomes (early endosomes, multivesicular bodies, late endosomes, and lysosomes) in the cytoplasm of mammalian cells, in which molecules are sorted for internalization through cell-mediated internalization processes, such as pinocytosis, micropinocytosis, receptor-mediated endocytosis, and / or phagocytosis.

[0122] Once endosomes in the endosomal / lysosomal pathway have been purified or isolated, assays for the target protein (e.g., an antigen-binding protein construct described herein) can be performed using methods known in the art (e.g., ELISA, Western blot, immunofluorescence, and immunoprecipitation followed by assays for protein concentration) and used to determine the concentration or relative levels of the target protein in the endosomes. Alternatively, endosomes in the endosomal / lysosomal pathway can be imaged using immunofluorescence microscopy using a detectably labeled antibody (e.g., a fluorescently, dye-, or GFP-labeled antibody, e.g., CellLight™ Early Endosome-GFP) that specifically binds to a characteristic protein present in endosomes (e.g., EEA1 for early endosomes) and a fluorescently labeled antibody (e.g., an antigen-binding protein construct) that specifically binds to the protein of interest, and the level of the target protein in the endosome can be determined by quantitating the overlap in fluorescence emission of the two different antibodies.

[0123] The phrase "endolysosomal delivery" refers to the rate of accumulation over time or the total amount of accumulation at a particular time point of an antigen binding protein construct (e.g., any of the antigen binding protein constructs described herein) in the endosomal / lysosomal pathway in a mammalian cell (e.g., any of the exemplary target mammalian cells described herein).

[0124] An exemplary method for calculating the increase in endolysosomal delivery of a pH-engineered ABPC variant compared to its corresponding starting ABPC from cytofluorescence data is to measure the ratio of the mean fluorescence intensity of the variant minus the mean fluorescence intensity of the unconjugated IgG control, and then divide the total by the mean fluorescence intensity of the variant's corresponding starting ABPC minus the mean fluorescence intensity of the IgG control.

[0125] Exemplary assays for measuring endolysosomal delivery of any of the ABPCs described herein include labeling the ABPC with a fluorescent dye, followed by incubating the labeled ABPC with cells and measuring cellular fluorescence as an indicator of endolysosomal delivery of ABPC (e.g., as generally described in Wustner, Traffic 7(6):699-715, 2006). Alternatively, any of the ABPCs described herein can be labeled with a pH-sensitive dye that preferentially fluoresces at acidic pH, but not at neutral pH, which can then be incubated with cells and cellular fluorescence measured as an indicator of delivery of ABPC into the acidic endolysosomal compartment.

[0126] The term "population" when used before a noun means two or more of that particular noun. For example, the phrase "population of cancer cells" means "two or more cancer cells." Non-limiting examples of cancer cells are described herein.

[0127] The phrase "cytostatic to a cell" refers to a direct or indirect reduction in proliferation (cell division) of a cell (e.g., a cancer cell) in vivo or in vitro. When an agent is cytostatic to a cell, the agent can, for example, directly or indirectly cause cell cycle arrest of the cell (e.g., a cancer cell). In some examples, an agent that is cytostatic to a cell can reduce the number of cells in a population of cells that are in S phase (compared to the number of cells in that population of cells that are in S phase prior to contact with the agent). In some examples, an agent that is cytostatic to a cell can reduce the percentage of cells in S phase by at least 20%, at least 40%, at least 60%, or at least 80% (e.g., compared to the percentage of cells in a population of cells that are in S phase prior to contact with the agent).

[0128] The phrase "cytotoxic to a cell" refers to the induction, directly or indirectly, of cell (eg, mammalian cell, eg, cancer cell) death (eg, necrosis or apoptosis).

[0129] "Affinity" refers to the strength of the sum total of 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 the intrinsic binding affinity, which reflects a 1:1 interaction between a member of the ABD and an antigen or epitope. The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ). 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) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining affinity for ABDs and their corresponding antigens or epitopes are known in the art.

[0130] The term "epitope" refers to the portion of an antigen that is specifically bound by an ABD through a series of physical interactions between: (i) all monomers (e.g., individual amino acid residues, sugar side chains, and post-translationally modified amino acid residues) on the portion of the ABD that specifically binds to the antigen; and (ii) all monomers (e.g., individual amino acid residues, sugar side chains, and post-translationally modified amino acid residues) on the portion of the antigen that is specifically bound by the ABD. An epitope may consist of, for example, surface-accessible amino acid residues, sugar side chains, phosphorylated amino acid residues, methylated amino acid residues, and / or acetylated amino acid residues, but may also have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, may be lost in the presence of denaturing solvents. 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 of a full-length protein or a portion thereof that is defined by three-dimensional structure (e.g., protein folding). In some embodiments, an epitope is defined by discontinuous amino acid sequences held together through protein folding. In some embodiments, an epitope is defined by discontinuous amino acid sequences held together by quaternary structure (e.g., a cleft formed by the interaction of two different polypeptide chains). The amino acid sequence between the residues that define the epitope may not be critical to the three-dimensional structure of the epitope. Conformational epitopes may be determined and screened using assays that compare the binding of antigen-binding protein constructs to denatured versions of the antigen, as linear epitopes are generated. Epitopes may include amino acid residues directly involved in the binding and other amino acid residues not directly involved in the binding.

[0131] Methods for identifying epitopes to which ABDs specifically bind are known in the art, such as structure-based analyses (e.g., X-ray crystallography, NMR, and / or electron microscopy) (e.g., on antigen and / or antigen-ABD complexes) and / or mutagenesis-based analyses in which variants are measured in binding assays with binding partners (e.g., alanine scanning mutagenesis, glycine scanning mutagenesis, and homology scanning mutagenesis), many of which are known in the art.

[0132] The term "paratope" refers to the portion of the ABD that specifically binds to an antigen through a series of physical interactions between: (i) all monomers (e.g., individual amino acid residues, sugar side chains, post-translationally modified amino acid residues) on the portion of the ABD that specifically binds to the antigen, and (ii) all monomers (e.g., individual amino acid residues, sugar side chains, post-translationally modified amino acid residues) on the portion of the antigen that is specifically bound by the ABD. A paratope can consist of, for example, surface-accessible amino acid residues, but can also have specific three-dimensional structural characteristics, as well as specific charge characteristics. In some embodiments, a paratope refers to a portion of a full-length ABD or a portion thereof that is defined by a three-dimensional structure (e.g., protein folding). In some embodiments, a paratope is defined by discontinuous amino acid sequences held together through protein folding. In some embodiments, an epitope is defined by discontinuous amino acid sequences held together by a quaternary structure (e.g., a cleft formed by the interaction of two different polypeptide chains). The amino acid sequence between the residues that define the paratope may not be critical to the three-dimensional structure of the paratope. The paratope can include amino acid residues that are directly involved in the bond and other amino acid residues that are not directly involved in the bond.

[0133] Methods for identifying the paratope to which an ABD specifically binds are known in the art, such as structure-based analyses (e.g., X-ray crystallography, NMR, and / or electron microscopy) (e.g., on the ABD and / or ABD-antigen complex), and / or mutagenesis-based analyses in which variants are measured in binding assays with a binding partner (e.g., alanine scanning mutagenesis, glycine scanning mutagenesis, and homology scanning mutagenesis), many of which are known in the art.

[0134] The phrase "present on the surface of a mammalian cell" refers to either (1) an antigen that is physically attached or at least partially embedded in the plasma membrane of a mammalian cell (e.g., a transmembrane protein, a peripheral membrane protein, a lipid-anchored protein (e.g., a GPI anchor), an N-myristolyated protein, or an S-palmitoylated protein), or (2) an antigen stably bound to its cognate receptor, where the cognate receptor is physically attached to the plasma membrane of a mammalian cell (e.g., a ligand bound to its cognate receptor, where the cognate receptor is physically attached to the plasma membrane). Non-limiting methods for determining the presence of an antigen on the surface of a mammalian cell include fluorescence-activated cell sorting (FACS), immunohistochemistry, cell fractionation assays, and Western blot.

[0135] The phrase "control ABPC" or "control antigen-binding protein construct" means an ABPC that (i) is capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed 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 ABD at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein) is greater than or equal to the dissociation rate of the first ABD at a pH of about 7.0 to about 8.0 (e.g., any of the subranges of this range described herein). or (b) a dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein); or (b) a dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein); D ) at a pH of about 7.0 to about 8.0 (e.g., any subrange of the range described herein), D is ≦3 times (e.g., ≦2.8, ≦2.6, ≦2.5, ≦2.4, ≦2.2, ≦2.0, ≦1.8, ≦1.6, ≦1.5, ≦1.4, ≦1.2, ≦1.0, ≦0.8, ≦0.6, ≦0.5, ≦0.4, ≦0.3, ≦0.2, or ≦0.1 times) greater than (i) 15G7; (ii) 24D9; and / or (iii) 29F1.

[0136] The term "extracellular space" refers to the fluid external to the plasma membrane of a mammalian cell. When the mammalian cell is in vitro, the extracellular space can be liquid culture medium. When the mammalian cell is in vivo, the extracellular space can be, for example, plasma, serum, blood, interstitial fluid, or lymph.

[0137] The term "endolysosomal space" refers to the fluid enclosed by the vesicles and organelles that make up the endosomal / lysosomal pathway in mammalian cells.

[0138] The phrase "decreased level" or "diminished level" can be a decrease or reduction of at least 1% (e.g., ≥ 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 ≥ 99%) as compared to a reference level or value.

[0139] The term "cell-killing ability" refers to the ability of an agent (e.g., any of the ABPCs described herein) to directly or indirectly induce apoptosis and / or necrosis in mammalian cells (e.g., cancer cells), measured as a rate over time or at relevant time points. Methods for determining the cell-killing ability of a cell are known in the art (e.g., trypan blue staining, microscopy, fluorescence-assisted cell sorting, and assays for detecting apoptotic markers (e.g., Annexin V)). In non-limiting examples, cell-killing ability can be measured, for example, by cell killing at a single concentration of the agent, by the agent's IC50 (i.e., the agent's concentration that achieves half of the maximum cell-killing ability), or by the ratio of the agent's dissociation constant KD in mammalian cells divided by its IC50. In some non-limiting examples, the IC50 and / or KD ratio described herein are compared to those of a control ABPC (as defined herein), and in some cases, demonstrate that the ABPC described herein has a higher cell-killing ability than the control ABPC.

[0140] The term "toxin release" refers to the ability of mammalian cells (e.g., non-cancerous mammalian cells or cancer cells) to internalize (e.g., via pinocytosis and / or receptor-mediated endocytosis) any of the ABPCs described herein (e.g., any of the ABPCs described herein or control ABPCs) conjugated to a toxin and subsequently release the toxin conjugated to the ABPC, measured as a percentage over time or at specific time points. Toxin release can be assessed using a variety of different exemplary assays, such as ELISA, immunofluorescence, cell killing assays, cell cycle arrest assays, DNA damage assays, mass spectrometry, HPLC, and / or isotope-labeled toxin.

[0141] The phrases "target cell" or "target mammalian cell" or "mammalian target cell" refer to a mammalian cell having at least one LRRC15 present on its surface. In some examples, the mammalian target cell can be a cancer cell. Some embodiments of target mammalian cells can have a total of about 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, 1-45,000 LRRC15 present on the plasma membrane of target mammalian cells in any of the ranges of values ​​recited in U.S. Patent Application No. 2022 / 0281984, which is incorporated by reference in its entirety.

[0142] The term "antigen density" refers to the number of LRRC15 present on the surface of a target mammalian cell, or the average number of LRRC15 on the surface of a population of target mammalian cells of a particular type, which can be measured, for example, using a QuantiBRITE™ bead kit or radiolabeling (e.g., BD Biosciences PE Phycoerythrin Fluorescence Quantitation Kit, catalog number 340495).

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

[0144] The phrase "amino acid substituted with alanine" refers to the substitution of an amino acid residue that is a histidine in a reference polypeptide sequence with alanine. Non-limiting methods for substituting alanine for histidine in a reference polypeptide are described herein. Additional methods for substituting alanine for histidine in a reference polypeptide are known in the art.

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0146] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawings]

[0147] [Figure 1A-1] Figure 1A shows the binding of 15G7 and its HC variants to LRRC15 by biolayer interferometry (BLI). For each individual plot, the antigen-binding protein was captured on an anti-human Fc biosensor and associated with LRRC15 at pH 7.4. Dissociation was at pH 7.4 (black) or pH 5.4 (gray). The major x-axis scale = 100 seconds, with the left dashed vertical line = 0 seconds. The major y-axis scale = 0.5 nm and octet plots are shown for MYT5712 and MYT6080-MYT6121. [Figure 1A-2] Same as above.

[0148] [Figure 1B] Figure 1B shows the binding of 15G7 and its LC variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown, MYT6122 to MYT6151.

[0149] [Figure 1C] Figure 1C shows the binding of humanized 15G7 and its HC variants to LRRC15 by BLI. Major Y-axis scale = 0.5 nm and octet plots are shown, MYT8387 to MYT8411.

[0150] [Figure 1D] Figure 1D shows the binding of humanized 15G7 and its LC variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown, MYT8413 to MYT8437.

[0151] [Figure 1E-1] FIG. 1E shows the binding data of FIG. 1A plotted to maximize vertical separation. [Figure 1E-2] Same as above.

[0152] [Figure 1F] FIG. 1F shows the binding data of FIG. 1B plotted to maximize vertical separation.

[0153] [Figure 1G] FIG. 1G shows the binding data of FIG. 1C plotted to maximize vertical separation.

[0154] [Figure 1H] FIG. 1H shows the binding data of FIG. 1D plotted to maximize vertical separation.

[0155] [Figure 2A-1] Figure 2A shows the binding of 24D9 and its HC variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown for MYT5742 and MYT7881 to MYT7925. [Figure 2A-2] Same as above.

[0156] [Figure 2B-1] FIG. 2B shows the binding data of FIG. 2A plotted to maximize vertical separation. [Figure 2B-2] Same as above.

[0157] [Figure 3A] Figure 3A shows the binding of 24D9 and its LC variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown for MYT7926 to MYT7958.

[0158] [Figure 3B] FIG. 3B shows the binding data of FIG. 3A plotted to maximize vertical separation.

[0159] [Figure 4A-1]Figure 4A shows the binding of humanized 29F1 and its HC aspartic acid variants to LRRC15 by BLI. Major Y-axis scale = 0.1 nm and octet plots are shown for MYT8094 and MYT9464-MYT9506. [Figure 4A-2] Same as above.

[0160] [Figure 4B-1] FIG. 4B shows the binding data of FIG. 4A plotted to maximize vertical separation. [Figure 4B-2] Same as above.

[0161] [Figure 5A] Figure 5A shows the binding of humanized 29F1 and its LC aspartic acid variants to LRRC15 by BLI. Major Y-axis scale = 0.1 nm and octet plots are shown for MYT9507 to MYT9534.

[0162] [Figure 5B] FIG. 5B shows the binding data of FIG. 5A plotted to maximize vertical separation.

[0163] [Figure 6A-1] Figure 6A shows the binding of humanized 29F1 and its HC glutamate variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown for MYT9684 to MYT9726. [Figure 6A-2] Same as above.

[0164] [Figure 6B-1] FIG. 6B shows the binding data of FIG. 6A plotted to maximize vertical separation. [Figure 6B-2] Same as above.

[0165] [Figure 7A]Figure 7A shows the binding of humanized 29F1 and its LC glutamate variants to LRRC15 by BLI. Major Y-axis scale = 0.2 nm and octet plots are shown for MYT9727 to MYT9754.

[0166] [Figure 7B] FIG. 7B shows the binding data of FIG. 7A plotted to maximize vertical separation.

[0167] [Figure 8] FIG. 8 shows a graph depicting the median tumor volume (TV) at the indicated days following administration of the indicated test substances or controls to SA4033 tumor-bearing mice.

[0168] [Figure 9] FIG. 9 presents a graph of median TV at the indicated days following administration of the indicated test substance or control in the CTG-0241 model.

[0169] [Figure 10A] FIG. 10A shows a graph of median TV on the indicated days following administration of the indicated test substance or control in the SA3851 model.

[0170] [Figure 10B] FIG. 10B shows a graph of median TV on the indicated days after administration of the indicated test substance or control in the SA4040 model.

[0171] [Figure 10C] FIG. 10C shows a graph of median TV on the indicated days after administration of the indicated test substance or control in the SA4109 model. DETAILED DESCRIPTION OF THE INVENTION

[0172] Provided herein is an antigen binding protein construct (ABPC) comprising a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, optionally 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 (K) of the first ABD at a pH of about 4.0 to about 6.5 is D ) at a pH of about 7.0 to about 8.0 D In some examples of these ABPCs, the ABPC is degraded in the target mammalian cell after internalization of the ABPC by the target mammalian cell. Some examples of any of the ABPCs described herein can further include a conjugated toxin, radioisotope, drug, or small molecule (e.g., a fluorophore or dye).

[0173] Also provided is an antigen binding protein construct (ABPC) comprising a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug, or small molecule, optionally comprising: (a) a dissociation rate of the first ABD at a pH of about 4.0 to about 6.5 that is faster than the dissociation rate at a pH of about 7.0 to about 8.0; and / or a dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5 that is faster than the dissociation rate at a pH of about 7.0 to about 8.0. D ) at a pH of about 7.0 to about 8.0 D and (b) the composition comprising ABPC provides one or more (e.g., two or three) of the following: an increase (e.g., a detectable increase) in toxin release in target mammalian cells compared to a composition comprising the same amount of control ABPC; an increase (e.g., a detectable increase) in target mammalian cell killing compared to a composition comprising the same amount of control ABPC; and an increase (e.g., a detectable increase) in endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of control ABPC.

[0174] In some examples, the first ABD comprises an HCV domain of 15G7, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the first ABD comprises an LCV domain of 15G7, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the first ABD comprises an HCV domain of 15G7, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine; and an LCV domain of 15G7, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the HCV domain of 15G7 comprises SEQ ID NO: 378 or 382. In some examples, the LCV domain of 15G7 comprises SEQ ID NO: 451 or SEQ ID NO: 455.

[0175] In some examples, the first ABD comprises an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379, 380, and 381, respectively, and optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 379-381 collectively substituted with histidine. In some examples, the first ABD comprises an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452, 453, and 454, respectively, and optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 452-454 collectively substituted with histidine. In some examples, the first ABD comprises an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 379, 380, and 381, respectively, and optionally collectively with all of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 379-381 substituted with histidine; and an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 452, 453, and 454, respectively, and optionally collectively with all of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 452-454 substituted with histidine.

[0176] In some examples, the first ABD comprises an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​and optionally the HCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO: 378 or 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110. In some examples, the first ABD comprises an LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, and optionally the LCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO: 451 or SEQ ID NO: 455. In some examples, the first ABD comprises an HCV domain that is ≥ 90% (e.g., ≥ 92%, ≥ 94%, ≥ 96%, ≥ 98%, ≥ 99%, or 100%) identical to the sequence set forth in SEQ ID NO: 378 or 382, ​​and optionally, the HCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110 in SEQ ID NO: 378 or 382. and an LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, and optionally the LCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97 in SEQ ID NO:451 or SEQ ID NO:455.

[0177] In some examples, the HCV domain includes an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​and the HCV domain includes a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1. [Table 1]

[0178] In some examples, the LCV domain includes an LCV domain that is ≥ 90% (e.g., ≥ 92%, ≥ 94%, ≥ 96%, ≥ 98%, ≥ 99%, or 100%) identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, and the LCV domain includes a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 451 or SEQ ID NO: 455 listed in Table 2. [Table 2]

[0179] In some examples, the first ABD comprises an HCV domain that is ≥90% identical (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1; and an LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence set forth in SEQ ID NO: 451 or 455, wherein the LCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 451 or 455 listed in Table 2.

[0180] In some examples, the first ABD includes an LCV domain comprising the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455 and an HCV domain that is ≧90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain includes a histidine at any of the specific combinations of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0181] In some examples, the first ABD comprises an LCV domain that is ≥90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises a histidine at position 29 in SEQ ID NO:451 or SEQ ID NO:455; and an HCV domain that is ≥90% identical (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) to the sequence set forth in SEQ ID NO:378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0182] In some examples, the first ABD comprises an LCV domain that is ≥90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises a histidine at position 30 in SEQ ID NO:451 or SEQ ID NO:455; and an HCV domain that is ≥90% identical (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) to the sequence set forth in SEQ ID NO:378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0183] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 32 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0184] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 34 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0185] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 50 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0186] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 92 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0187] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 93 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0188] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 95 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0189] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 96 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0190] In some examples, the first ABD comprises an LCV domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises a histidine at position 97 in SEQ ID NO: 451 or SEQ ID NO: 455; and an HCV domain that is at least 90% identical (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) to the sequence set forth in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 378 or 382 listed in Table 1.

[0191] In some examples, the first ABD comprises an HCV domain comprising a sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0192] In some examples, the first ABD comprises an LCV domain comprising a sequence set forth in one of SEQ ID NO: 451, SEQ ID NO: 455, or SEQ ID NOs: 456-511.

[0193] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 378 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0194] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 382 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0195] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 383 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0196] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 384 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0197] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 385 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0198] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 386 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0199] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 387 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0200] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 388 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0201] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 389 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0202] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 390 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0203] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 391 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0204] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 392 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0205] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 393 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0206] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 394 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0207] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 395 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0208] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 396 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0209] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 397 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0210] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 398 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0211] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 399 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0212] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 400 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0213] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 401 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0214] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 402 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0215] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 403 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0216] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 404 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0217] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 405 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0218] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 406 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0219] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 407 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0220] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 408 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0221] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 409 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0222] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 410 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0223] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 411 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0224] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 412 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0225] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 413 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0226] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 414 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0227] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 415 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0228] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 416 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0229] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 417 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0230] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 418 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0231] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 419 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0232] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 420 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0233] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 421 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0234] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 422 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0235] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 423 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0236] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 424 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0237] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 425 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0238] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 426 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0239] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 427 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0240] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 428 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0241] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 429 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0242] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 430 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0243] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 431 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0244] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 432 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0245] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 433 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0246] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 434 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0247] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 435 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0248] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 436 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0249] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 437 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0250] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 438 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0251] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 439 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0252] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 440 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0253] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 441 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0254] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 442 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0255] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 443 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0256] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 444 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0257] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 445 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0258] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 446 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0259] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 447 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0260] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 448 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0261] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 449 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0262] In some examples, the first ABD comprises an HCV domain comprising the sequence set forth in SEQ ID NO: 450 and an LCV domain comprising the sequence set forth in SEQ ID NO: 451, SEQ ID NO: 455, or one of SEQ ID NOs: 456-511.

[0263] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 451 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0264] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 456 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0265] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 457 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0266] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 458 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0267] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 459 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0268] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 460 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0269] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 461 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0270] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 462 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0271] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 463 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0272] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 464 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0273] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 465 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0274] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 466 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0275] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 467 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0276] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 468 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0277] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 469 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0278] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 470 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0279] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 471 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0280] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 472 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0281] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 473 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0282] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 474 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0283] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 475 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0284] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 476 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0285] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 477 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0286] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 478 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0287] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 479 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0288] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 480 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0289] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 481 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0290] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 482 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0291] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 483 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0292] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 484 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0293] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 485 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0294] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 486 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0295] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 487 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0296] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 488 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0297] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 489 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0298] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 490 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0299] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 491 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0300] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 492 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0301] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 493 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0302] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 494 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0303] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 495 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0304] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 496 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0305] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 497 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0306] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 498 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0307] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 499 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0308] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 500 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0309] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 501 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0310] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 502 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0311] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 503 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0312] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 504 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0313] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 505 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0314] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 506 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0315] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 507 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0316] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 508 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0317] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 509 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0318] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 510 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0319] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 511 and an HCV domain comprising the sequence set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​or one of SEQ ID NOs: 383-450.

[0320] In some examples, the first ABD comprises an HCV domain of 24D9, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the first ABD comprises an LCV domain of 24D9, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the first ABD comprises an HCV domain of 24D9, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine, and an LCV domain of 24D9, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine. In some examples, the HCV domain of 24D9 comprises the amino acid sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516. In some examples, the LCV domain of 24D9 comprises the amino acid sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522.

[0321] In some examples, the first ABD comprises an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513, 514, and 515, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine. In some examples, the first ABD comprises an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519, 520, and 521, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 519-521 collectively substituted with histidine. In some examples, the first ABD is an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 513, 514, and 515, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 513-515 collectively substituted with histidine; and an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 519, 520, and 521, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 519-521 collectively substituted with histidine.

[0322] In some examples, the first ABD comprises an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, and optionally the HCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO:512 or SEQ ID NO:516 selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110. In some examples, the first ABD comprises an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, and optionally the LCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO: 518 or SEQ ID NO: 522 selected from the group of 35 and 97. In some examples, the first ABD is an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO:512, and optionally the HCV domain is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110 in SEQ ID NO:512 or SEQ ID NO:516. and an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein optionally the LCV domain comprises a histidine at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of 35 and 97 in SEQ ID NO: 518 or SEQ ID NO: 522.

[0323] In some examples, the HCV domain includes an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516, and the HCV domain includes a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 512 or SEQ ID NO: 516 listed in Table 3. [Table 3]

[0324] In some examples, the LCV domain comprises an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, and the LCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 518 or SEQ ID NO: 522 listed in Table 4. [Table 4]

[0325] In some examples, the first ABD comprises an HCV domain comprising the sequence of SEQ ID NO: 512, SEQ ID NO: 516, or one of SEQ ID NOs: 523-571; and / or the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522.

[0326] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, and an HCV domain comprising the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516 or one of SEQ ID NOs: 523-567.

[0327] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, and an HCV domain comprising the sequence set forth in SEQ ID NO: 516 or one of SEQ ID NOs: 568-571.

[0328] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:512.

[0329] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:516.

[0330] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:523.

[0331] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:524.

[0332] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:525.

[0333] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:526.

[0334] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:527.

[0335] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:528.

[0336] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:529.

[0337] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:530.

[0338] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:531.

[0339] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:532.

[0340] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:533.

[0341] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:534.

[0342] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:535.

[0343] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:536.

[0344] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:537.

[0345] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:538.

[0346] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:539.

[0347] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:540.

[0348] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:541.

[0349] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:542.

[0350] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:543.

[0351] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:544.

[0352] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:545.

[0353] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:546.

[0354] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:547.

[0355] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:548.

[0356] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:549.

[0357] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:550.

[0358] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:551.

[0359] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:552.

[0360] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:553.

[0361] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:554.

[0362] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:555.

[0363] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:556.

[0364] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:557.

[0365] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:558.

[0366] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:559.

[0367] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:560.

[0368] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:561.

[0369] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:562.

[0370] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:563.

[0371] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:564.

[0372] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:565.

[0373] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:566.

[0374] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising the sequence of SEQ ID NO:567.

[0375] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:512.

[0376] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:516.

[0377] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:568.

[0378] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:569.

[0379] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:570.

[0380] In some examples, the first ABD comprises an LCV domain comprising the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, and an HCV domain comprising SEQ ID NO:571.

[0381] In some examples, the first ABD comprises the HCV domain of 29F1, optionally with one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0382] In some examples, the first ABD comprises the LCV domain of 29F1, optionally with one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0383] In some examples, the first ABD comprises an HCV domain of 29F1, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine, aspartic acid, or glutamic acid; and an LCV domain of 29F1, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0384] In some examples, the HCV domain of 29F1 comprises SEQ ID NO:572 or SEQ ID NO:572.

[0385] In some examples, the LCV domain of 29F1 comprises SEQ ID NO:577 or SEQ ID NO:581.

[0386] In some examples, the first ABD comprises an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 154, 155, and 156, respectively, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0387] In some examples, the first ABD comprises an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 157, 158, and 159, respectively, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 578-580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0388] In some examples, the first ABD includes an HCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 154, 155, and 156, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 573-575 collectively substituted with histidine, aspartic acid, or glutamic acid; and an LCV domain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 157, 158, and 159, respectively, optionally with a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NOs: 578-580 collectively substituted with histidine, aspartic acid, or glutamic acid.

[0389] In some examples, the first ABD comprises an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, and optionally the HCV domain comprises histidine, aspartic acid, or glutamic acid at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO: 572 or SEQ ID NO: 576 selected from the group of D positions selected from 31, 56, and 99; and E positions selected from 59. In some examples, the first ABD comprises an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, and optionally the LCV domain comprises histidine, aspartic acid (D), or glutamic acid (E) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions in SEQ ID NO: 577 or SEQ ID NO: 581 selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; or E positions selected from 51 and 56.In some examples, the first ABD is an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, and optionally the HCV domain comprises a histidine, aspartic acid, or glutamic acid at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of 27, 32, 33, 34, 35, 51, 54, 56, 58, and 100 in SEQ ID NO: 572 or SEQ ID NO: 576; and an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, and optionally the LCV domain comprises a histidine, aspartic acid (D), or glutamic acid (E) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid positions selected from the group consisting of D positions selected from 27, 28, 31, 52, and 56; or E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO: 581.

[0390] In some examples, the HCV domain includes an HCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, and the HCV domain includes a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 572 or SEQ ID NO: 576 listed in Table 5. [Table 5]

[0391] In some examples, the LCV domain comprises an LCV domain that is at least 90% (e.g., ≧92%, ≧94%, ≧96%, ≧98%, ≧99%, or 100%) identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, and the LCV domain comprises a histidine at any of the specific combinations of one or more amino acid positions in SEQ ID NO: 577 or SEQ ID NO: 581 listed in Table 6. [Table 6]

[0392] In some examples, the first ABD comprises an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695, and / or the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 577, SEQ ID NO: 581, one of SEQ ID NOs: 625-652, or one of SEQ ID NOs: 696-723.

[0393] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 577 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0394] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 581 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0395] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 625 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0396] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 626 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0397] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 627 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0398] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 628 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0399] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 629 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0400] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 630 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0401] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 631 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0402] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 632 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0403] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 633 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0404] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 634 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0405] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 635 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0406] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 636 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0407] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 637 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0408] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 638 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0409] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 639 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0410] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 640 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0411] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 641 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0412] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 642 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0413] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 643 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0414] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 644 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0415] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 645 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0416] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 646 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0417] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 647 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0418] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 648 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0419] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 649 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0420] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 650 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0421] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 651 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0422] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 652 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0423] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 696 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0424] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 697 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0425] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 698 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0426] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 699 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0427] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 700 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0428] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 701 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0429] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 702 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0430] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 703 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0431] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 704 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0432] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 705 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0433] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 706 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0434] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 707 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0435] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 708 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0436] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 709 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0437] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 710 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0438] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 711 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0439] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 712 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0440] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 713 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0441] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 714 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0442] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 715 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0443] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 716 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0444] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 717 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0445] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 718 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0446] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 719 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0447] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 720 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0448] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 721 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0449] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 722 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0450] In some examples, the first ABD comprises an LCV domain comprising the sequence of SEQ ID NO: 723 and an HCV domain comprising the sequence of SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, or one of SEQ ID NOs: 653-695.

[0451] Also provided herein are pharmaceutical compositions comprising any of the ABPCs described herein.

[0452] Also provided herein is a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the ABPCs described herein.

[0453] In some examples, a composition comprising an ABPC (e.g., any of the ABPCs described herein) can provide an increase (e.g., a detectable increase) in toxin release in a target mammalian cell (e.g., any of the target mammalian cells described herein) compared to a composition comprising the same amount of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., any of the percent increase or range of percent increase listed on pages 389-404 of WO2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984)).

[0454] In some examples, a composition comprising an ABPC (e.g., any of the ABPCs described herein) can provide an increase (e.g., a detectable increase) in toxin release in a target mammalian cell (e.g., any of the target mammalian cells described herein) compared to a composition comprising the same amount of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., any of the fold increase or range of fold increase listed on pages 404-420 of WO2021 / 022039 (or corresponding U.S. Patent Application No. 2022 / 0281984)).

[0455] In some examples, a composition comprising ABPC (e.g., any ABPC described herein) may result in an increase (e.g., a detectable increase) (e.g., ≥ 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 300%, 310%, 320%, 330%, 340%, 355%, 360%, 370%, 375%, 380%, 385%, 400%, 450%, 400%, 450%, 450%, 450%, 450%, 460%, 470%, 475%, 480%, 485%, 500%, 550%, 550%, 550%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 950%, 950%, 950%, 95 %, 90%, 95%, 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 a 1% to 10,000% increase).

[0456] In some examples, a composition comprising ABPC (e.g., any of the ABPCs described herein) exhibits an increase (e.g., a detectable increase) in target mammalian cell killing (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, 2.6, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10 5, 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 fold increase, or at least a 100 fold increase, or a 0.1 to 100 fold increase).

[0457] In some instances, a composition comprising any of the ABPCs described herein (e.g., upon contact with target mammalian cells displaying LRRC15 on their surface) exhibits an IC 50reduced (e.g., ≥ 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% reduced, or at least 99% reduced, about 1% to 99% reduced, or any subrange of this range described herein) IC compared to 50 (for target mammalian cell killing).

[0458] In some examples, a composition comprising any of the ABPCs described herein (e.g., upon contact with target mammalian cells that display LRRC15 on their surface) exhibits an increase (e.g., an IC at neutral pH on the same target cells) compared to, e.g., a control ABPC. 50 on target mammalian cells displaying LRRC15 on their surface at neutral pH Dcan provide at least a 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, or 95-fold increase, or at least a 100-fold increase, or about a 0.1 to 500-fold increase (or any subrange of this range described herein) in the ratio of In some examples, a composition comprising ABPC (e.g., any of the ABPCs described herein) may result in an increase (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%, 10%, 1 ...10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, An increase of 0%, 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 at least a 10,000% increase, or about a 1% to 10,000% increase (e.g., or any of the subranges of this range described herein) can be provided. In some examples, a composition comprising ABPC exhibits an increase (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, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.1, 11. 0.0, 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-fold increase, or at least a 100-fold increase, or about a 0.1 to 100-fold increase).In some examples of the ABPCs described herein, the target mammalian cells do not express FcRn receptors or express FcRn receptors at lower (e.g., detectably lower) levels (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%, or at least 99% reduction levels) compared to FcRn-expressing cells (e.g., HUVEC-ThermoFisher #C0035C). In some examples, the target mammalian cells are cancer cells. In some examples, the ABPC is cytotoxic or cytostatic to the target mammalian cells. In some examples, a composition comprising any of the ABPCs described herein (e.g., upon administration to a subject) results in a smaller reduction (e.g., a 1% reduction to about a 99% reduction, or any subrange within this range described herein) in the level of LRRC15 displayed on the surface of target cells compared to a composition comprising the same amount of a control ABPC (e.g., any of the control ABPCs described herein). In some examples, the composition does not result in a detectable reduction in the level of LRRC15 displayed on the surface of target mammalian cells. In some examples, ABPC is cross-reactive with non-human primate LRRC15 and human LRRC15. In some examples, ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, and one or both of rat LRRC15 and mouse LRRC15. In some examples, ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15. In some examples, ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15. In some examples, ABPC is cross-reactive with mouse LRRC15 and rat LRRC15. In some examples, the ABD binds to an epitope of LRRC15 present on the surface of cells from Old World monkeys. Some examples of any of the ABPCs 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 can be used in any combination without limitation. Additional embodiments of these methods are known in the art.

[0459] LRRC15 or an epitope of LRRC15 Several LRRC15-binding monoclonal antibodies have been described in the literature and can be used as templates for engineering pH-dependent binding. The amino acid sequence of mature human LRRC15 can be found in SEQ ID NO: 9. The amino acid sequence of the extracellular domain of LRRC15 can be found in SEQ ID NO: 11.

[0460] Antigen-binding protein constructs Any of the antigen binding protein constructs (ABPCs) described herein can be a single polypeptide or can comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 (the same or different) polypeptides. In some embodiments where the ABPC is a single polypeptide, the ABPC can comprise a single ABD or two ABDs. In some embodiments where the ABPC is a single polypeptide and comprises two ABDs, the first and second ABDs can be the same or different from each other (and can specifically bind to the same or different antigens or epitopes).

[0461] In some embodiments where the ABPC is a single polypeptide, the first ABD and second ABD (if present) can each be independently selected from the group of a VH domain, a VHH domain, a VNAR domain, and an scFv. In some embodiments where the ABPC is a single polypeptide, the antigen binding protein construct can be a BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, sc diabody, sc diabody-CH3, scFv-CH-CL-scFv, HSAbody, sc diabody-HAS, tandem-scFv, adnectin, DARPin, fibronectin, and DEP conjugate.

[0462] In some embodiments, the ABPC or ABD comprises a proteolysis-targeting antibody (PROTAB) (e.g., as described in Marei et al., "Antibody targeting of E3 ubiquitin ligases for receptor degradation." Nature, October 6, 2022). In some embodiments, the PROTAB or PROTAC can tether a cell surface E3 ubiquitin ligase to a transmembrane protein, resulting in targeted degradation both in vitro and in vivo.

[0463] Additional examples of ABDs that can be used when the ABPC is a single polypeptide are known in the art.

[0464] V H The H domain is a single monomeric variable antibody domain that can be found in camelids. NAR The V domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. H H domain and V NARNon-limiting embodiments of domains are described, for example, in 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. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0465] In some embodiments where the ABPC is a single polypeptide and comprises two ABDs, the first ABD and the second ABD can both be VHH domains, or at least one ABD can be a VHH domain. In some embodiments where the ABPC is a single polypeptide and comprises two ABDs, the first ABD and the second ABD can both be VHH domains.NAR domain, or at least one ABD is V NAR In some embodiments where the ABPC is a single polypeptide, the first ABD is an scFv domain. In some embodiments where the ABPC is a single polypeptide and comprises two ABDs, the first ABD and the second ABD can both be scFv domains, or at least one ABD can be an scFv domain.

[0466] In some embodiments, the ABPC can comprise two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides). In some embodiments in which the ABPC comprises two or more polypeptides, 2, 3, 4, 5, or 6 of the two or more polypeptides can be identical.

[0467] In some embodiments, an ABPC comprises two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides), two or more of the polypeptides of the ABPC are associated (non-covalently associated) to form one or more ABDs, such as an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), a VHH-scAb, a VHH-Fab, a double scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-in-one), a DAF (fo ur-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knobs-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, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, dock and lock, ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, and DEP conjugates can be formed. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which is incorporated herein in its entirety for a description of these elements.

[0468] Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments. Additional examples of antigen-binding fragments of antibodies 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, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2); 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).

[0469] An "Fv" fragment comprises a non-covalently linked dimer of one HCV and one LCV domain.

[0470] The "Fab" fragment contains the heavy chain domain and LCV domain of the Fv fragment as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ) is included.

[0471] An "F(ab')2" fragment contains two Fab fragments joined near the hinge region by a disulfide bond.

[0472] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to a multivalent and multispecific binding protein 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. Patent Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

[0473] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0474] Additional embodiments of ABPC are known in the art.

[0475] antigen-binding domain In some embodiments of any of the antigen binding protein constructs (ABPCs) described herein, the dissociation rate of the first ABD (and optionally, the second ABD, if present) is a dissociation rate at a pH of about 7.0 to about 8.0 (e.g., about 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). , 7.1~7.6, 7.1~7.5, 7.1~7.4, 7.1~7.3, 7.1~7.2, 7.2~8.0, 7.2~7.9, 7.2~7.8, 7.2~7.7, 7.2~7.6, 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, a pH of about 4.0 to about 6.5 (e.g., about 4.0 to about 6.4, or those listed on pages 428-430 of WO2021 / 022039 (or corresponding U.S. patent application Ser. No. 09 / 02203924)) rather than 7.5-8.0, 7.5-7.9, 7.5-7.8, 7.5-7.7, 7.5-7.6, 7.379-381.0, 7.6-7.9, 7.6-7.8, 7.6-7.7, 7.7-8.0, 7.7-7.9, 7.7-7.8, 7.8-8.0, 7.8-7.9, or about 7.9-8.0). WO 2021 / 022039, pages 430-447 (or corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entireties), fast (e.g., at least 5% fast, or any of the % fast, or % range fast listed in WO 2021 / 022039, pages 430-447 (or corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entireties).

[0476] In some embodiments of any 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 within this range described herein) is D ), is greater (e.g., detectably greater) (e.g., greater than K at a pH of about 7.0 to about 8.0 (e.g., any of the subranges of this range described herein) D More than 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% greater, or about 5% to about 10,000% greater, or greater than those listed on pages 448-466 of WO2021 / 022039 (or those listed in corresponding U.S. Patent Application No. 2022 / 0281984). D (The range is any of the percentages of the total amount of the hydroxybenzoates, which are incorporated herein by reference).

[0477] In some embodiments of any 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 (e.g., at least 0.2-fold faster, or any of the fold faster or fold range faster listed on pages 466-468 of WO2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated by reference in their entireties herein) 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 described herein).

[0478] In some embodiments of any 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 within this range described herein) is D ) is the K at a pH of about 7.0 to about 8.0 (e.g., any of the subranges of this range described herein). D is greater (e.g., detectably greater) than (e.g., at least 0.2 times greater, or any of the fold or range of fold greater listed on pages 468-470 of WO2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated by reference herein in their entireties).

[0479] In some embodiments of an ABPC comprising a first ABD and a second ABD, the first and second ABD are identical or at least 80% identical in amino acid sequence to each other (e.g., ≧82%, ≧84%, ≧86%, ≧88%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% identical). In some embodiments, for an ABPC comprising a first ABD and a second ABD, the first ABD and 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% identical). In some embodiments of an ABPC comprising a first and second ABD, the first and second ABD bind to two different epitopes (e.g., two different epitopes on LRRC15, or a first ABD that specifically binds to LRRC15 and a second ABD that binds to an antigen other than LRRC15).

[0480] In some embodiments of any of the ABPCs described herein, the K of the first ABD (and, optionally, the second ABD, if present) at a pH of about 7.0 to 8.0 (e.g., any of the subranges of this range described herein) is D is about 1 pM to 5 μM (e.g., 1 pM to 2 μM, or as listed on pages 471 to 492 of WO2021 / 022039 (or as listed in corresponding U.S. Patent Application No. 2022 / 0281984) D and the like, which are incorporated by reference herein in their entireties).

[0481] In some embodiments of any of the ABPCs 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 subranges of this range described herein) is D is 1 nM (e.g., between about 1 nM and 1 mM, or as listed on pages 492-496 of WO2021 / 022039 (or as listed in corresponding U.S. Patent Application No. 2022 / 0281984) D and the like, which are incorporated by reference herein in their entireties.

[0482] A variety of different methods known in the art can be used to cleave any of the antigen binding protein constructs described herein. D The antibody can be used to determine the binding activity (e.g., electrophoretic mobility shift assay, filter binding assay, surface plasmon resonance, biomolecular binding kinetics assay, in vitro binding assay on antigen-expressing cells, etc.).

[0483] In some examples, the half-life of ABPC in vivo is decreased (e.g., detectably decreased) compared to the half-life of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., at least a 1% decrease, or any of the percent decreases or ranges of percent decreases listed on pages 496-500 of WO2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated by reference herein in their entireties). Conversely, in some examples, the half-life of ABPC in vivo may be increased compared to the half-life of the control ABPC.

[0484] Conjugates In some embodiments, the ABPC provided herein can be conjugated to a drug (e.g., a chemotherapy drug, a small molecule), a toxin, or a radioisotope. Non-limiting examples of drugs, toxins, and radioisotopes are known in the art.

[0485] 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 comprises a protease cleavage site. In some embodiments, the cleavable linker is cleaved on the ABPC once it is transported by the target mammalian cell to a lysosome or late endosome. In some embodiments, cleavage of the linker functionally activates the drug or toxin.

[0486] 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, hi some embodiments, the conjugated toxin, radioisotope, or drug is released during lysosomal and / or late endosomal degradation of the ABPC.

[0487] Non-limiting examples of cleavable linkers include hydrazone linkers, peptide linkers, disulfide linkers, and thioether linkers. See, e.g., 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.

[0488] Non-limiting examples of non-cleavable linkers include maleimidoalkane linkers and maleimidocyclohexane linkers (MMC) (see, e.g., those described in McCombs et al., AAPS J. 17(2):339-351, 2015).

[0489] In some embodiments, any of the ABPCs described herein are cytotoxic or cytostatic to target mammalian cells.

[0490] In some embodiments, the antibodies provided herein can include one or more amino acid substitutions to provide a conjugation site (e.g., conjugated to a drug, toxin, or radioisotope). In some embodiments, the antibodies provided herein can have one conjugation site. In some embodiments, the antibodies described herein can have two conjugation sites. In some embodiments, the antibodies provided herein can have three or more conjugation sites. Non-limiting examples of amino acid substitutions that generate conjugation sites (e.g., "triple hinge" conjugation sites) are described in U.S. Patent Application Publication No. 2017 / 0348429, which is incorporated herein by reference in its entirety. For example, a lysine to cysteine ​​substitution at amino acid position 105 of SEQ ID NO: 351 or SEQ ID NO: 352 and deletion of the threonines at amino acid positions 106 and 108 can provide a "triple hinge" conjugation site in any of the antibodies described herein. In some embodiments, an alanine to cysteine ​​substitution at amino acid position 1 of SEQ ID NO: 351 or SEQ ID NO: 352 can provide a conjugation site for any of the antibodies described herein. In some embodiments, a Val to Cys substitution at position 98 of SEQ ID NO: 353 can provide a conjugation site for any of the antibodies described herein.

[0491] The native cysteine ​​amino acid can also be provided conjugated (e.g., conjugated to a drug, toxin, radioisotope). In some embodiments, the antibodies provided herein can have a drug, toxin, or radioisotope conjugated at one or more (e.g., one, two, three, or four) native conjugation sites. In some embodiments, the cysteine ​​at amino acid position 103 of SEQ ID NO: 351 or 352 is a native conjugation site. In some embodiments, the cysteine ​​at amino acid position 109 of SEQ ID NO: 351 or 352 is a native conjugation site. In some embodiments, the cysteine ​​at amino acid position 112 of SEQ ID NO: 5 or 6 is a native conjugation site. In some embodiments, the cysteine ​​at amino acid position 107 of SEQ ID NO: 353 is a native conjugation site.

[0492] In some embodiments, the antibodies provided herein can have a drug, toxin, or radioisotope conjugated at one or more (e.g., two, three, or four) native conjugation sites, e.g., the cysteine ​​at amino acid position 103, the cysteine ​​at amino acid position 109, and / or the cysteine ​​at amino acid position 112, and / or the cysteine ​​at amino acid position 107 of SEQ ID NO: 353, of SEQ ID NO: 351 or SEQ ID NO: 352. In some embodiments, the antibodies provided herein can have a drug, toxin, or radioisotope conjugated at one or more native conjugation sites and one or more engineered conjugation sites.

[0493] Conjugation via engineered cysteines is achieved by methods known in the art. Briefly, engineered cysteine-containing antibodies are prepared for conjugation 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 disrupts interchain disulfides with disulfide bonds in the antibody and removes the disulfide cap from the engineered cysteine. An optional reoxidation step, achieved by exposing the solution to air or an oxidizing agent, such as dehydroascorbic acid, allows the interchain disulfide bonds to reform, leaving the engineered cysteine ​​with a thiolate-reactive group. Conjugation with the maleimide functionality on the linker-payload maleimide-vc-MMAE is achieved by reaction with the payload in a buffered 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 selective filtration methods such as tangential flow filtration, which removes residual unreacted payload, reducing agent, and oxidizing agent from the reaction mixture, and the conjugated ADC product may be transferred into a desired formulation buffer.

[0494] Conjugation through hinge cysteines is achieved by similar methods using antibodies with or without additional engineered cysteine ​​conjugation sites. Briefly, the antibody is prepared for conjugation by treatment with a reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT). The reducing agent's reducing strength and concentration are selected so that some or all of the interchain disulfide bonds are reduced, leaving free cysteines for conjugation. Solutions may also be directly conjugated in the presence of excess reducing agent. Conjugation with the maleimide functionality on the linker-payload maleimide-vc-MMAE is achieved by reaction with the payload in a buffered solution containing a cosolvent, such as ethanol, dimethylacetamide (DMA), or dimethyl sulfoxide (DMSO). Unreacted linker-payloads can be rendered unreactive by the addition of a sacrificial thiolate molecule, such as acetyl-cysteine. The crude conjugated antibody solution can be further purified by methods known in the art, including hydrophobic interaction chromatography, ion exchange chromatography, or mixed-mode chromatography, such as ceramic hydroxyapatite chromatography. Isolation of chromatographic fractions allows for selection of the desired antibody to payload ratio and removal of unreacted antibody, protein aggregates and fragments, and payload-related reaction by-products. The purified antibody-drug conjugate can be further purified by size exclusion chromatography or selective filtration methods, such as TFF. During this step, the conjugated ADC product can also be transferred into a desired formulation buffer.

[0495] In some examples, antibody conjugates can be made containing an antibody linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereafter referred to as LRRC15-IgG-DC). Conjugation of the antibody with vcMMAE begins with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (10 mg / mL) is partially reduced by the addition of TCEP (TCEP:mAb molar equivalent ratio is 2:1), followed by overnight incubation at 4°C. The reduction reaction is then warmed to 25°C. To conjugate all of the thiols, vcMMAE is added to a final vcMMAE:reduced Cys molar ratio of 1:10. The conjugation reaction was carried out in the presence of 10% v / v dimethylacetamide (DMA) and allowed to proceed for 60 minutes at 25°C.

[0496] In some examples, antibody conjugates (ADCs) are prepared comprising the LRRC15-binding IgG (hereinafter, LRRC15-IgG) described herein linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereinafter, LRRC15-IgG-DC). Conjugation of the antibody with vcMMAE begins with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (10 mg / mL) is reduced by the addition of DTT (the molar equivalent of DTT:mAb is 100:1), followed by overnight incubation at 25°C. The reduced LRRC15-IgG (10 mg / mL) is then reoxidized by exposure to DHAA (the molar equivalent of DHAA:mAb is 10:1), followed by incubation for 2 hours at 25°C. To conjugate all of the thiols, vcMMAE is added to a final vcMMAE:mAb molar ratio of 4: 1. The conjugation reaction is carried out in the presence of 10% v / v DMA and allowed to proceed for 3 hours at 25°C.

[0497] Expression of antigen-binding protein constructs in cells Also provided herein are methods for producing a recombinant cell that expresses ABPC (e.g., any of the ABPCs described herein), comprising introducing a nucleic acid encoding ABPC into a cell to produce the recombinant cell; and culturing the recombinant cell under conditions sufficient for expression of ABPC. In some embodiments, the introducing step comprises introducing an expression vector comprising a nucleic acid encoding ABPC into the cell to produce the recombinant cell.

[0498] Any of the ABPCs described herein can be produced by any cell, e.g., a eukaryotic cell or a prokaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell with a separate, membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, e.g., Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic organism, e.g., a mammalian, avian, plant, or insect cell. As used herein, the term "prokaryotic cell" refers to a cell that does not have a separate, membrane-bound nucleus. In some embodiments, the prokaryotic cell is a bacterial cell.

[0499] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor proliferation, differentiation, and growth. Briefly, cells can be cultured by contacting the cells (e.g., any cell) with a cell culture medium containing the necessary growth factors and supplements to support cell viability and growth.

[0500] Methods for introducing nucleic acids and expression vectors into cells (e.g., eukaryotic cells) are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetic transfer, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

[0501] Provided herein are methods that further include isolating ABPC from cells (e.g., eukaryotic cells) using techniques well known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion exchange chromatography (anionic or cationic), hydrophobic interaction-based chromatography, metal affinity chromatography, ligand affinity chromatography, and size exclusion chromatography).

[0502] Avidity Antibodies and antigen-binding fragments thereof are multivalent and thus contain more than one binding site. A measure of the total binding strength of an antibody at its binding site is generally referred to as avidity. Generally, the terms "fold avidity" and "selectivity" can refer to the fold difference between an antibody's affinity and its avidity, as seen, for example, when measuring the total binding strength of an antibody on a cell line with high target expression (avidity; e.g., cancer cells, e.g., SAOS-2 cells) compared to a cell line with low target expression (affinity; e.g., non-cancer cells, e.g., G-292 cells). Generally, avidity is determined by four factors: binding affinity (e.g., strength of binding at individual binding sites); valency (e.g., total number of binding sites); structural configuration (e.g., antigen and antibody structure); and antigen density (e.g., number of antigens per cell).

[0503] Provided herein are methods for reducing the risk of developing metastases or reducing the risk of developing additional metastases in a subject having cancer, wherein the cancer is characterized by having a population of cancer cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, the method comprising administering a therapeutically effective amount of any of the antibodies described herein or any of the pharmaceutical compositions described herein to a subject identified as having a cancer characterized by having a population of cancer cells. In some embodiments, the antibodies described herein exhibit at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or more selectivity for cancer cells compared to non-cancerous cells. In some embodiments, the cancer is a sarcoma, e.g., leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0504] Treatment methods Provided herein are methods of treating cancer characterized by having a population of cancer cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, 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 a cancer characterized by having a population of cancer cells.

[0505] Also provided herein are methods for reducing tumor volume in a subject, wherein the tumor is characterized by having a population of cancer cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, comprising administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPC described herein to a subject identified as having a cancer characterized by having a population of cancer cells. In some embodiments of any of the methods described herein, the volume of at least one tumor or tumor site is reduced by ≧1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% compared to the size of the at least one tumor before administration of ABPC.

[0506] Also provided herein are methods of inducing cell death of cancer cells in a subject, the methods 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 a cancer characterized as having a population of cancer cells, the cancer cells having LRRC15 or an epitope of LRRC15 displayed on their surface. In some embodiments, the induced cell death is necrosis or apoptosis.

[0507] In some embodiments of any of the methods described herein, the cancer is a primary tumor. In some embodiments of any of the methods described herein, the cancer is a metastasis. In some embodiments of any of the methods described herein, the cancer is a non-T cell infiltrating tumor. In some embodiments of any of the methods described herein, the cancer is a T cell infiltrating tumor. In some embodiments of any of the methods described herein, the cellular compartment is part of the endosomal / lysosomal pathway. In some embodiments of any of the methods described herein, the cellular compartment is an endosome. In some embodiments, the cancer is a sarcoma, such as leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0508] Provided herein are methods for reducing the risk of developing metastases or additional metastases in a subject with cancer, wherein the cancer is characterized by having a population of cancer cells that have LRRC15 or an epitope of LRRC15 displayed on their surface, 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 a cancer characterized as having a population of cancer cells. In some embodiments, the risk of developing metastases or additional metastases in the subject is reduced by at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% compared to the risk in a subject with a similar cancer but administered no treatment or a treatment that does not include administration of any of the ABPCs described herein.

[0509] The term "subject" refers to any mammal. In some embodiments, the subject or "subject suitable for treatment" can be a canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), ovine, bovine, porcine, caprine, primate, such as a primate (e.g., monkey (e.g., marmoset, baboon), or ape (e.g., gorilla, chimpanzee, orangutan, or gibbon) or human; or a rodent (e.g., mouse, guinea pig, hamster, or rat). In some embodiments, the subject or "subject suitable for treatment" can be a non-human mammal, but in particular, a mammal (e.g., murine, lapine, porcine, canine, or primate) traditionally used as a model for demonstrating therapeutic efficacy in humans can be used.

[0510] As used herein, treating includes reducing the number, frequency, or severity of one or more (e.g., two, three, four, or five) signs or symptoms of cancer in a patient with cancer (e.g., any of the cancers described herein). For example, treating can reduce the progression of cancer, reduce the severity of cancer, or reduce the risk of cancer recurrence in a subject with cancer.

[0511] Provided herein is a method of inhibiting the growth of a solid tumor in a subject (e.g., any of the subjects described herein) (e.g., compared to the growth of the solid tumor in the subject before treatment, or compared to the growth of a similar solid tumor in a different subject that has received a different treatment or has not received treatment), comprising administering to the subject a therapeutically effective amount of any of the ABPCs described herein or any of the pharmaceutical compositions described herein.

[0512] In some embodiments of any of the methods described herein, the solid tumor growth is primary growth of a solid tumor. In some embodiments of any of the methods described herein, the solid tumor growth is recurrent growth of a solid tumor. In some embodiments of any of the methods described herein, the solid tumor growth is metastatic growth of a solid tumor. In some embodiments, the treatment results in about a 1% to about a 99% reduction (or any subrange of this range described herein) in solid tumor growth in the subject (e.g., compared to the growth of the solid tumor in the subject before treatment, or compared to the growth of a similar solid tumor in a different subject that has received a different treatment or no treatment). Solid tumor growth in a subject can be assessed by a variety of different imaging methods, such as positron emission tomography, x-ray computed tomography, computed tomography, and magnetic resonance imaging.

[0513] Also provided herein are methods of reducing the risk of developing metastases or additional metastases over a period of time in a subject identified as having cancer (e.g., any of the exemplary cancers described herein) (e.g., compared to a subject with the same cancer who is receiving a different treatment or no treatment), comprising administering to the subject a therapeutically effective amount of any of the proteins described herein or any of the pharmaceutical compositions described herein. In some embodiments of any of the methods described herein, the metastases or additional metastases are to one or more of bone, lymph nodes, brain, lung, liver, skin, chest wall including bone, cartilage and soft tissue, abdominal cavity, contralateral breast, soft tissue, muscle, bone marrow, ovaries, adrenal glands, and pancreas.

[0514] In some embodiments of any of the methods described herein, the period of time is from about 1 month to about 3 years (e.g., about 1-30, 1-24, 2-18, 1-12, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-36, 2-30, 2-24, 2-18, 2-12, 2-10, 2-8, 2-6, 2-5, 2-4, 2-3, 3-36, 3-30, 3-24, 3-18, 3-12, 3-10, 3-8, 3-6, 379-381, 3-4, 4-36, 4-30, 4-24, 4-18, 4-12, 4- 10, 4-8, 4-6, 4-5, 5-36, 5-30, 5-24, 5-18, 5-12, 5-10, 5-8, 5-6, 6-36, 6-30, 6-24, 6-18, 6-12, 6-10, 379-381, 8-36, 8-30, 8-24, 8-18, 8-12, 8-10, 10-36, 10-30, 10-24, 10-18, 10-12, 12-36, 12-30, 12-24, 12-18, 18-36, 18-30, 18-24, 24-36, 24-30 months, or about 30-36 months).

[0515] In some embodiments, the risk of developing metastases or additional metastases over a period of time in a subject identified as having cancer is reduced by, e.g., about 1% to about 99% (e.g., or any of the subranges within this range described herein) compared to the risk in a subject with a similar cancer who is receiving a different treatment or no treatment.

[0516] Non-limiting examples of cancer include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, uterine leukemia, and thyroid cancer. Langerhans sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary, midline tract carcinoma involving NUT gene gene), oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. Additional examples of cancers are known in the art.

[0517] In some embodiments, the patient is further administered one or more additional therapeutic agents (e.g., one or more of a chemotherapeutic agent, a recombinant cytokine or interleukin protein, a kinase inhibitor, and a checkpoint inhibitor). In some embodiments, the one or more additional therapeutic agents are administered to the patient at about the same time that any of the ABPCs described herein are administered to the patient. In some embodiments, the one or more additional therapeutic agents are administered to the patient after administration of any of the ABPCs described herein to the patient. In some embodiments, the one or more additional therapeutic agents are administered to the patient before administration of any of the ABPCs described herein to the patient.

[0518] In some embodiments of any of the methods described herein, the cancer is a solid cancer (e.g., breast cancer, prostate cancer, or non-small cell lung cancer). In some embodiments, the cancer is a sarcoma, such as leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0519] composition Also provided herein are compositions (e.g., pharmaceutical compositions) comprising at least one of any of the ABPCs described herein. In some embodiments, the compositions (e.g., pharmaceutical compositions) can be disposed in sterile vials or pre-filled syringes.

[0520] In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, or intratumor). In some embodiments, the composition (e.g., pharmaceutical composition) can include a pharmaceutically acceptable carrier (e.g., phosphate-buffered saline). Single or multiple administrations of any of the pharmaceutical compositions described herein can be given to a subject, depending, for example, on the dosage and frequency required and tolerated by the patient. The dosage of the pharmaceutical composition should provide a sufficient amount of ABPC to effectively treat or ameliorate a condition, disease, or symptom.

[0521] Also provided herein are methods of treating a subject having cancer (e.g., any of the cancers described herein), comprising administering a therapeutically effective amount of at least one of any of the compositions or pharmaceutical compositions provided herein.

[0522] kit Also provided herein are kits comprising any of the ABPCs described herein, any of the compositions described herein, or any of the pharmaceutical compositions described herein. In some embodiments, the kits can include instructions for carrying out any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0523] Protein constructs Also provided is a protein construct (PC) comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD at a pH of about 7.0 to about 8.0 (or any subrange of this range described herein) is faster than the dissociation rate at a pH of about 4.0 to about 6.5 (or any subrange of this range described herein); and / or (b) the dissociation constant (K) of the first ABD at a pH of about 7.0 to about 8.0 (or any subrange of this range) is greater than or equal to 1.0; D ) at a pH of about 4.0 to about 6.5 D Greater than.

[0524] Also provided herein are pharmaceutical compositions comprising any of the PCs described herein. Also provided are methods of treating a subject in need thereof, comprising administering a therapeutically effective amount of any of the PCs described herein.

[0525] Methods for improving the pH dependence of antigen-binding protein constructs Also provided herein is a method of improving the pH dependency of an antigen binding protein construct, the method comprising providing a starting antigen binding protein construct comprising an ABD, and introducing one or more histidine amino acid substitutions into one or more CDRs of the ABD in the starting antigen binding protein construct, wherein the method results in: (a) an increased ratio of the dissociation rate of the ABD at a pH of about 4.0 to about 6.5 to the dissociation rate at a pH of about 7.0 to about 8.0, compared to the starting antigen binding protein construct (e.g., at least a 0.1-fold increase to about a 100-fold increase, or any subrange of this range described herein); and (b) an increased dissociation constant (K D ) at a pH of about 7.0 to about 8.0 D or both (e.g., at least a 0.1-fold increase to about a 100-fold increase, or any of the subranges within this range described herein).

[0526] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0527] Example 1. Generation of LRRC15-binding agents and manipulation of pH-binding dependence pH-engineered ABPCs specific for LRRC15 can be generated using several methods. In the first approach, a publicly available or de novo identified monoclonal antibody against LRRC15 can be used as a starting template for introducing mutations, thereby enabling the engineering of pH-dependent binding to LRRC15, resulting in i) enhanced endolysosomal accumulation of conjugated toxins, and ii) enhanced LRRC15 recycling to the cell surface. The second approach involves the discovery of novel ABPCs specific for LRRC15 through antibody display methods from naive libraries or libraries with defined CDR compositions and screening under conditions designed to select pH-engineered ABPCs specific for LRRC15. In each case, histidine residues play a key role in engineering pH-dependent binding proteins.

[0528] Due to its pKa of 6.0, histidine residues are at least partially protonated below pH 6.5. Thus, if a histidine side chain in an antigen-binding domain is involved in an electrostatic binding interaction with the antigen, it will begin to acquire a positive charge at or below pH 6.5. This can either weaken or enhance the binding affinity of the interaction at pH below 6.5, depending on the corresponding charge of the antigen epitope and its interaction with the antigen epitope. Thus, systematic introduction of histidines into antibody complementarity-determining regions (CDRs) in a library of antibodies or other binders (e.g., an scFv library) can be used to identify substitutions that may affect the interaction of the antigen-binding domain with the antigen at lower pH values. A first approach, therefore, involves histidine scanning of published monoclonal antibody variable domain sequences to identify pH-dependent variants.

[0529] Several LRRC15-binding monoclonal antibodies have been described in the literature and can be used as templates for engineering pH-dependent binding, Purcell et al., LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates, Cancer Res. 78(14):4059-4072 (2018). Briefly, for a subset of antibody sequences, the CDRs in each chain were identified using the methods described by Kabat et al. (Kabat et al. (1992) Sequences of Proteins of Immunological Interest (DIANE Publishing)) and IMGT (Lefranc MP (1999) "The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domains," The Immunologist 7, 132-136), and for each CDR, residues that fit either or both of the Kabat and IMGT CDR definitions are designated as CDR residues. To engineer pH-dependent sequence variants, individual amino acid residues within the heavy and / or light chain CDRs are systematically substituted with histidine, one at a time. If the starting CDR residue is histidine, it is mutated to alanine. Antibody variants with only one histidine or alanine mutation in the heavy / light chain CDR are generated by co-transfection of Expi293 cells with a) one heavy or light chain sequence variant and b) the corresponding light or heavy chain, respectively, of the starting ABPC (e.g., the starting LRRC15-binding monoclonal antibody), using methods known in the art.

[0530] After allowing protein expression for a period of time, cell culture supernatants are collected and quantified, and the pH dependence of the variants is assessed using biolayer interferometry (BLI) or other methods known in the art. Briefly, cell culture supernatants are normalized to an antibody expression level of 50 μg / mL and captured on an anti-human Fc sensor (Forte Bio). A baseline is established using 1X kinetics buffer (Forte Bio), and the sensor is then associated with 100 nM LRRC15 in 1X PBS at pH 7.4 for 300 seconds to generate an association curve. During the dissociation phase, the antibody-antigen complex on the sensor is exposed to 1X PBS at either pH 5.5 or pH 7.4 for 300-500 seconds. Association and dissociation curves are examined for the starting ABPC antibody and each corresponding antibody variant at pH 5.5 and pH 7.4 to obtain information on two criteria: a) enhanced dissociation at pH 5.5 (i.e., higher koff values) due to histidine or alanine substitutions compared to the starting ABPC, and b) decreased dissociation at pH 7.4 (i.e., lower koff values) compared to pH 5.5, both for the antibody variant itself and for the starting ABPC. Variants showing either enhanced dissociation at pH 5.5 or decreased dissociation at pH 7.4 are selected for further analysis. It is also noted that some histidine and alanine mutations abolish LRRC15 binding, while others are tolerated and associated with minor or no changes in LRRC15 binding kinetics (e.g., less than a 1-fold change in KD or dissociation rate). In particular, because histidine is a large, positively charged amino acid, these unchanged histidine and alanine variants are noted as positions that tolerate a wide range of mutations and can lead to antibodies with different sequences but similar binding properties, otherwise unclear nomenclature. Variants selected for further analysis are expressed on a larger scale and purified using protein A affinity chromatography. The binding kinetics (k and k) of the purified starting ABPC and variant antibodies are measured at pH 5.5 and pH 7.4 using Biacore (GE Healthcare).The ratio of antibody dissociation rates (koff at pH 7.4 divided by koff at pH 5.5) is also used as a quantitative assessment of pH-dependent binding; similarly, the dissociation constant KD is calculated as koff divided by kon at both pH 5.5 and pH 7.4, and the ratio of antibody dissociation constants (KD at pH 7.4 divided by KD at pH 5.5) is also used as a quantitative assessment of pH-dependent binding.

[0531] Antibodies with dissociation rate ratios and / or dissociation constant ratios that are lower than those of the starting ABPC are selected for further evaluation of combinatorial substitutions.Preferred histidine and / or alanine amino acid positions can also be combined to enhance pH dependency; for example, by combining or rationally combining histidine and / or alanine substitutions that individually improve pH dependency on a given heavy or light chain, for example, by combining or rationally combining modified heavy and light chains, so that histidine and / or alanine substitutions are present on both chains, or combinations thereof.Such combinatorial variants are generated and tested / analyzed for differential pH dependency using the methods and protocols described herein or others known in the art.The antibody variant with the lowest dissociation rate ratio and / or dissociation constant ratio is selected as a candidate for further analysis (hereinafter referred to as "LRRC15-specific pH-engineered ABPC").

[0532] A second method for selecting pH-engineered ABPCs specific for LRRC15 involves screening libraries to identify either novel pH-dependent ABPCs specific for LRRC15 or ABPCs that may serve as templates for engineering pH-dependent binding as described herein. Two types of libraries can be used for these selections: naive phage / yeast display antibody libraries (e.g., Fab, scFv, VHH, VL, or others known in the art), or phage / yeast display libraries in which CDRs have been mutated to express a subset of amino acid residues. The libraries are screened against soluble recombinant LRRC15 extracellular domain using methods known in the art, with positive selection for variants that bind weakly at pH 5.0 (e.g., eluted from beads) and strongly at pH 7.4 (e.g., bound to beads). Three rounds of selection are performed. Final round binders are screened for binding to human LRRC15, cynomolgus monkey LRRC15, and mouse LRRC15 using ELISA or via mean fluorescence intensity in flow cytometry analysis. If more binders with cynomolgus monkey or mouse cross-reactivity are desired, the final selection round can be performed with cynomolgus monkey LRRC15 or mouse LRRC15 instead. Selected binding proteins are subcloned into mammalian expression vectors and expressed in Expi293 cells as either full IgG proteins or Fc fusions. BLI analysis is performed as described herein for the selection of pH-dependent binder variants and confirmed using Biacore.

[0533] Production of 15G7, 24D9, and 29F1 anti-LRRC15 monoclonal antibodies Applicant inoculated rabbits with the extracellular domain of the human LRRC15 protein to generate 250 rabbit mAbs (RabMAbs). After initial generation of b-cell clones, cell supernatants were evaluated in secondary cytotoxicity assays. Each Ab that killed the cells was sequenced, procured, expressed, and re-assayed for cytotoxicity, internalization, and other properties as a chimeric antibody (i.e., rabbit variable domain with human constant region). Top candidates were humanized, and in parallel, histidine, aspartic acid, glutamic acid, and other amino acid scanning variants of the chimeric antibodies were produced as described in U.S. Patent Application No. US 2022 / 0281984, which is incorporated herein by reference in its entirety.

[0534] Once promising RabMAb mutations and humanized frameworks were identified, the mutations were transferred to the humanized framework, and the resulting humanized mutant antibodies were retested for activity and developability. The most promising humanized mutant Abs were then conjugated with a toxic payload (see, e.g., paragraphs [0495-0496] above) to generate candidate anti-LRRC15 ADCs, which were then evaluated for stability, activity, and developability. The higher-performing candidates are listed in Table 7, and additional details are provided in Example 23 (below). [Table 7-1] [Table 7-2]

[0535] Tables 8-13 provide details about constructs of particular utility during the humanization process. And, while the development candidates disclosed in Table 7 (above) are among the most advanced, many useful intermediate forms have been produced in applicants' efforts to improve the developability of the binding proteins disclosed herein. Developability modifications include those that tend to improve Ab solubility, reduce aggregation, and optimize in vivo half-life. Applicants have fully disclosed the various "developability modifications" that are permissible and / or beneficial, and one of skill in the art may routinely produce similar binding proteins with desired drug-like properties. The table immediately below introduces these developability constructs and some of their associated properties. [Table 8] [Table 9] [Table 10] [Table 11-1] [Table 11-2] [Table 12] [Table 13-1] [Table 13-2] [Table 14-1] [Table 14-2] [Table 14-3]

[0536] Example 2. In vitro demonstration of pH-dependent binding to LRRC15, pH-dependent release of LRRC15, enhanced endolysosomal delivery in LRRC15+ cells, and increased LRRC15 antigen density in LRRC15+ cells after exposure to pH-engineered ABPC specific for LRRC15 compared to control ABPC specific for LRRC15 As discussed herein, pH-engineered ABPCs specific for LRRC15 exhibit the desirable properties of reduced LRRC15 binding at acidic pH (e.g., pH 5.0 to pH 6.4) but enhanced binding at higher, e.g., physiological pH (e.g., pH 7.4), thereby enhancing their accumulation in endolysosomes under physiological conditions.

[0537] pH-dependent binding to LRRC15 on cells To demonstrate that the pH-engineered ABPC specific for LRRC15 binds to cell surface LRRC15 at neutral pH, a cell surface binding assay is performed. A panel of LRRC15+ human cells is assembled. Methods for identifying and quantifying gene expression (e.g., LRRC15) for a given cell line are known in the art and include, for example, consulting the Cancer Cell Line Encyclopedia (CCLE; https: / / portals.broadinstitute.org / ccle) to confirm the expression level and / or mutation status of a given gene in tumor cell lines, rtPCR, microarrays, RNA-Seq analysis, or cell staining with antibodies known in the art (e.g., recombinant anti-LRRC15 antibody, Abcam catalog number ab150376 clone EPR8188(2); Hi-Affi™ recombinant rabbit anti-LRRC15 monoclonal antibody, Creative Biolabs catalog number MOR-2090 Clone#DS2090AB for LRRC15). Cells were seeded at approximately 5-10,000 cells per well in 150 μL of pH 7.4 medium and incubated for 5 minutes at 37°C with one of the following antibodies at several doses (e.g., 2-fold dilutions) ranging from 1 pM to 1 μM: a known control ABPC specific for LRRC15 (e.g., antibody samrotamab, hu139.10, huAD208.4.1, huAD208.12.1, 1-13C3, or 1-19G12), a pH-engineered ABPC specific for LRRC15, and a suitable negative isotype control mAb (e.g., Biolegend purified human IgG1 isotype control recombinant antibody, catalog no. 403501). Prior to initiation of the experiment, the binding properties of all antibodies were verified using methods known in the art. After a 5-minute incubation, cells are fixed with 4% formaldehyde (20 minutes at room temperature) and incubated with a suitable fluorophore-conjugated secondary antibody (e.g., ThermoFisher Mouse Anti-Human IgG1 Fc Secondary Antibody, Alexa Fluor 488, Cat. No. A-10631) for 60 minutes. Unbound reagents are washed away with a series of PBS washes, and cell panels are imaged using a confocal microscope.Upon image analysis, significant fluorescence was observed on the surface of cells bound with known, control ABPCs specific for LRRC15, as well as pH-engineered ABPCs specific for LRRC15, while minimal surface binding was observed for the isotype-negative control. To isolate the effect of pH on surface binding, the same experiment was repeated twice, with primary antibody incubation occurring at successively lower pHs (e.g., pH 6.5, pH 5.5, and pH 5.0). Analysis of the resulting confocal microscopy images showed significant fluorescence on the surface of cells bound with all tested mAbs, except for the isotype-negative control, which decreased as the pH decreased for the pH-engineered ABPCs specific for LRRC15. Alternatively, cells were analyzed for mean fluorescence intensity by flow cytometry using methods known in the art. The dissociation constants (KD) of the analyzed antibodies on cells at neutral pH, KD, were determined by nonlinear regression methods known in the art (e.g., Scatchard plots). Taken together, these results demonstrate that the pH engineering process results in the creation of a pH-engineered ABPC specific for LRRC15 that is pH-dependent in its binding properties and binds more effectively at neutral pH compared to more acidic pH. Other methods for assessing the pH dependence of pH-engineered ABPCs specific for LRRC15 are known in the art, for example, measuring ABPC surface binding using flow cytometry.

[0538] pH-dependent release of LRRC15 on cells To demonstrate that the pH-engineered ABPC specific for LRRC15 can bind at neutral pH and then release LRRC15 at low pH, a variant of the cell surface binding assay described above is performed using methods known in the art (e.g., as generally described in Gera N. (2012) PLoS ONE 7(11):e48928). Briefly, suitable LRRC15+ cell lines (less than 25 passages) are harvested and 50,000 cells per well are seeded into a U-bottom 96-well microplate. Three conditions are tested: binding and secondary staining at pH 7.4, binding and secondary staining at pH 5.0, and binding at pH 7.4 followed by release at pH 5.0 for 30 minutes and secondary staining at pH 7.4. Both the pH-engineered ABPC specific for LRRC15 and a control ABPC specific for LRRC15 are tested. Cells are washed twice with 200 μL of FACS buffer (1× PBS containing 3% fetal bovine serum) at either pH 7.4 or 5.0, depending on the conditions being tested. Purified protein samples are diluted in FACS buffer at the appropriate pH and added to the cells for binding on ice for 1 hour. After incubation with the primary antibody, the cells are washed twice as before for pH 7.4 and pH 5.0 conditions, and then 100 μL of a 1:50 dilution of secondary rat anti-human FcAF488 (BioLegend 410706) or other suitable antibody, or a 1:50 dilution of anti-Myc-Tag mouse mAb-AF488 (Cell Signaling Technologies 2279S) is added in FACS buffer at the appropriate pH and incubated on ice for 30 minutes. The pH 5.0 release condition is washed twice with FACS buffer pH 7.4, then resuspended in 100 μL of FACS buffer pH 5.0 and incubated on ice for 30 minutes, followed by secondary staining in FACS buffer pH 7.4 as described for other conditions. The plate is washed twice as before and resuspended in 1% paraformaldehyde in the appropriate FACS buffer to fix for flow cytometry analysis. All conditions are read on a flow cytometer (Accuri C6, BD Biosciences).Binding is observed as a shift in the F1 signal (as mean fluorescence intensity) relative to the second order alone. Upon analysis of the data, it can be determined that both the pH-engineered ABPC specific for LRRC15 and the control ABPC specific for LRRC15 effectively bind to the surface of LRRC15+ cells at neutral pH, but that the pH-engineered ABPC specific for LRRC15 binds poorly at pH 5.0; similarly, it can be determined that the pH-engineered ABPC specific for LRRC15 binds at pH 7.4, but then releases LRRC15 at pH 5.0.

[0539] Enhanced endolysosomal delivery of pH-engineered ABPC specific for LRRC15 in LRRC15+ cells compared to control ABPC specific for LRRC15 To verify and demonstrate that LRRC15-specific ABPCs achieve endolysosomal localization after cellular uptake, internalization assays are performed using methods known in the art (e.g., Mahmutefendic et al., Int. J. Biochem. Cell Bio., 2011). Briefly, as described herein, a panel of human cells highly expressing LRRC15 is assembled using methods known in the art. Cells are seeded, washed three times with PBS, and incubated with a known LRRC15-specific control ABPC (e.g., as described herein), a pH-manipulated ABPC specific for LRRC15, and an appropriate negative isotype control mAb (e.g., as described herein) at a concentration of 2 micrograms / milliliter in medium at neutral pH for 60 minutes at 37°C. Verification of antibody internalization and endosomal localization in a subset of cells is performed using methods known in the art; for example, cells are fixed in 4% formaldehyde as described herein, permeabilized using TWEEN® 20 or other methods known in the art (Jamur MC et al. (2010) Permeabilization of cell membranes, Methods Mol Biol. 588:63-6), and additionally stained with an endosomal marker, e.g., fluorescent RAB11 antibody (RAB11 antibody, Alexa Fluor 488, 3H18L5, ABfinity™ rabbit monoclonal antibody), stained with a suitable fluorescently labeled anti-human secondary antibody (e.g., as described herein), and imaged using a confocal fluorescence microscope as described herein. Analysis of confocal images can be used to show that both the pH-engineered ABPC specific for LRRC15, as well as the control ABPC specific for LRRC15, are internalized and accumulate in endolysosomes.

[0540] To demonstrate that a pH-engineered ABPC specific for LRRC15 achieves enhanced endolysosomal accumulation compared to a control ABPC specific for LRRC15, a pHrodo-based internalization assay was performed using both a known control ABPC specific for LRRC15 (e.g., as described herein) and a pH-engineered ABPC specific for LRRC15. The assay used pHrodo™ iFL (P36014, ThermoFisher), a dye whose fluorescence increases as the pH decreases, such that the level of extracellular fluorescence at neutral pH is lower than the level of fluorescence within the acidic pH environment of the endolysosome. Briefly, a suitable LRRC15+ cell line (<25 passages) was suspended in its recommended medium (e.g., according to a cell bank or cell bank database, ATCC, DSMZ, or ExPASy Cellosaurus) and seeded into a 24-well plate at a density of 2,000,000 cells / mL, 1 mL per well. While the cells were kept on ice, 1 mL of 2x pHrodo iFL-labeled antibody (prepared according to the manufacturer's instructions) was added to each well, the wells were pipetted / mixed five times, and the plate was incubated on ice in a light-protected environment for 45 minutes. An identical but separate plate was also incubated on ice. This served as a negative control for non-internalization. After this incubation, the experimental plate was transferred to a 37°C incubator, and the negative control plate was kept on ice to delay or block internalization. Samples were taken at designated time points to create a time course of internalization. Samples were plated into a U-bottom 96-well plate, and internalization was quenched by adding 200 μL / well of ice-cold FACS buffer. The plate was spun down at 2000×g for 2 minutes, resuspended in 200 μL of ice-cold FACS buffer, spun down again, and resuspended a second time in FACS buffer.Finally, the sample was loaded into a flow cytometer for cellular pHrodo fluorescence readings using excitation and emission wavelengths that matched the excitation and emission maxima of the pHrodo iFL red dye (566 nm and 590 nm, respectively). Upon completion of the flow cytometry experiment and data analysis, it could be observed that cells treated with the pH-engineered ABPC specific for LRRC15 had a higher pHrodo iFL signal than the known, control ABPC specific for LRRC15, indicating that the pH-engineered ABPC specific for LRRC15 achieved enhanced endolysosomal accumulation compared to the control ABPC specific for LRRC15.

[0541] Alternatively, to demonstrate that pH-engineered ABPC specific for LRRC15 achieves enhanced endolysosomal accumulation compared to control ABPC specific for LRRC15, a variation of the experiment described above was performed. LRRC15+ cells were seeded, washed three times with PBS, and incubated with either pH-engineered ABPC specific for LRRC15 or control ABPC specific for LRRC15 at a concentration of 2 μg / mL in medium at neutral pH for 60 minutes at 37°C. After incubation, the cells were washed three times with PBS, fixed and permeabilized, and stained with a panel of appropriately selected antibodies that bind to late endosome markers and lysosomes (e.g., RAB7 and LAMP1; Cell Signaling Technology, Endosomal Marker Antibody Sampler Kit No. 12666; AbCam, anti-LAMP2 antibody [GL2A7], ab13524). After primary antibody staining, cells are stained with an appropriate mixture of fluorescently labeled secondary antibodies (e.g., goat anti-human IgG (H&L) secondary antibody (Alexa Fluor 647), catalog number A-21445, and Abcam goat anti-rabbit IgG H&L (Alexa Fluor 488), catalog number ab150077) and imaged using a confocal fluorescence microscope to visualize and quantify the area of ​​colocalization of signals from the LRRC15-specific antibody and endosomal markers. Analysis of the data can reveal increased colocalization of endolysosomal and LRRC15-specific antibody signals in wells treated with the pH-manipulated ABPC specific for LRRC15 compared to wells treated with the control ABPC specific for LRRC15, thereby demonstrating that the pH-manipulated ABPC specific for LRRC15 achieves enhanced endolysosomal accumulation compared to the control ABPC.

[0542] Increased LRRC15 antigen density in LRRC15+ cells after exposure to pH-engineered ABPC specific for LRRC15 compared to control ABPC specific for LRRC15To demonstrate that treatment of cells with a pH-engineered ABPC specific for LRRC15 does not result in a detectable reduction in the level of LRRC15 on the surface of cells exposed to the pH-engineered ABPC specific for LRRC15, or that the treatment results in a lesser reduction in the level of LRRC15 on the surface of cells exposed to the pH-engineered ABPC specific for LRRC15 relative to the control ABPC specific for LRRC15, an antigen density test is performed using flow cytometry. Briefly, 4.0 x 10^5 cells expressing LRRC15 are seeded in 100 μL of medium per well in a 96-well plate. Cells are treated with increasing doses ranging from 1 pM to 1 μM of i) the pH-engineered ABPC specific for LRRC15, ii) a first control ABPC specific for LRRC15, iii) an appropriate isotype control, and iv) an untreated control. The cells are incubated for 2 hours at 37°C, at which point all cells are incubated for 30 minutes at 4°C with 200 nM of a fluorophore-labeled second control ABPC specific for LRRC15 (e.g., as described herein), which has an epitope different from either the first control ABPC specific for LRRC15 or the pH-manipulated ABPC specific for LRRC15 (e.g., as determined by competitive binding assays on cells). After this 30-minute incubation, the mean fluorescence intensity (MFI) of all cells is read using methods known to those skilled in the art, for example, using flow cytometry. In parallel, a quantitative standard curve that can be used to quantify the presence of LRRC15 on the surface of treated cells as a function of MFI is generated using a commercially available quantification kit (e.g., BD Biosciences PE Phycoerythrin Fluorescence Quantification Kit, catalog number 340495); the quantitative standard curve is prepared according to the manufacturer's instructions. Other methods for determining the absolute number of cell surface LRRC15 are known in the art and include, for example, the use of radioisotope-labeled reagents.Upon analysis of the data, it may become apparent that, at least at one antibody concentration, cells treated with a control ABPC specific for LRRC15 experience a reduction in the levels of LRRC15 on their surface, whereas cells treated with a pH-engineered ABPC specific for LRRC15 experience a significantly smaller reduction or no reduction at all compared to both the isotype control and the untreated control.

[0543] Example 3. Conjugation of pH-engineered and control ABPC to cytotoxic drugs An antigen-binding protein construct conjugate (ADC) is generated containing the LRRC15-binding IgG described herein (hereafter LRRC15-IgG) linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereafter LRRC15-IgG-DC). Conjugation of the antigen-binding protein construct with vcMMAE begins with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (20 mg / mL) is partially reduced by the addition of TCEP (TCEP:mAb molar equivalents are 2:1), followed by overnight incubation at 0°C. The reduction reaction is then warmed to 20°C. To conjugate all of the thiols, vcMMAE is added to a final vcMMAE:reduced Cys molar ratio of 1:15. The conjugation reaction was carried out in the presence of 10% v / v DMSO and allowed to proceed for 60 min at 20°C.

[0544] After the conjugation reaction, excess free N(acetyl)-cysteine ​​(2 equivalents relative to the vcMMAE charge) is added to quench unreacted vcMMAE to produce the Cys-Val-Cit-MMAE adduct. The Cys-quenching reaction is allowed to proceed at 20°C for approximately 30 minutes. The Cys-quenching reaction mixture is purified as follows. The above conjugation method can also be used to conjugate maleimidocaproyl monomethyl auristatin F (mcMMAF) to an antigen-binding protein construct.

[0545] LRRC15-IgG-DC is purified using a batch purification method. The reaction mixture is treated with an appropriate amount of water-washed Bu-HIC resin (ToyoPearl; Tosoh Biosciences)—i.e., seven weights of resin are added to the mixture. The resin / reaction mixture is stirred for an appropriate time, monitored by analytical hydrophobic interaction chromatography for removal of the drug conjugate product, filtered through a coarse polypropylene filter, and washed with two bed volumes of buffer (0.28 M sodium chloride, 7 mM potassium phosphate, pH 7). The combined filtrate and rinse are combined and analyzed for product profile by HIC HPLC. The combined filtrate and rinse are buffer exchanged by ultrafiltration / diafiltration (UF / DF) with 10 diavolumes of 15 nM histidine buffer to 15 mM histidine, pH 6.

[0546] Similar protocols can be used to conjugate DNA toxins, such as SG3249 and SGD-1910, to LRRC15-IgG (see Tiberghien AC et al. (2016) Design and Synthesis of Tesirine, a Clinical Antibody-Drug Conjugate Pyrrolobenzodiazepine Dimer Payload, ACS Med Chem Lett 7:983-987). Briefly, for SG3249, LRRC15-IgG (15 mg, 100 nmoles) was diluted in 13.5 mL of reduction buffer containing 10 mM sodium borate pH 8.4 and 2.5 mM EDTA, for a final antibody concentration of 1.11 mg / mL. A 10 mM solution of TCEP was added (1.5 molar equivalents / antibody, 150 nmoles, 15 microliters), and the reduction mixture was heated to +37°C for 1.5 hours in an incubator. After cooling to room temperature, SG3249 is added as a DMSO solution (5 molar equivalents / antibody, 500 nanomoles in 1.5 mL of DMSO). The solution is mixed at room temperature for 1.25 hours, and then the conjugate is quenched by adding N-acetylcysteine ​​(1 micromolar, 100 microliters at 10 mM) and injected into an AKTA™ Pure FPLC using a GE Healthcare HiLoad™ 26 / 600 column packed with Superdex 200PG and eluted with sterile-filtered phosphate-buffered saline (PBS) at 2.6 mL / min. The fractions corresponding to the LRRC15-IgG-DC monomer peak are pooled, concentrated using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, analyzed, and sterile-filtered.UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150x2.1mm column eluted with a gradient of water and acetonitrile at 280nm and 330nm (SG3249 specific) for a reduced sample of LRRC15-IgG-DC could show a mixture of light and heavy chains attached to several molecules of SG3249, consistent with a drug-antibody ratio (DAR) of 1-4 molecules of SG3249 per antibody. UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Yarra 3u SEC-3000 300 mm x 4.60 mm column shows a monomeric purity of greater than 90% at 280 nm for a sample of LRRC15-IgG-DC eluted with a sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v). UHPLC SEC analysis allows the determination of a final LRRC15-IgG-DC yield of greater than 30%.

[0547] Alternatively, methods for conjugating toxins to antibodies via lysine residues are known in the art (see, e.g., Catcott KC et al. (2016) Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates, MAbs 8:513-23). ​​Similar methods can also be used to conjugate drugs and toxins to non-IgG formats involving disulfide bonds, such as Vh-Fc.

[0548] Example 4. Demonstration of enhanced cytotoxicity of pH-engineered ABPC ADC specific for LRRC15 in LRRC15+ cells compared to control ABPC ADC specific for LRRC15 The cytotoxic activity of both the pH-engineered ADC specific for LRRC15 (e.g., pH-engineered LRRC15-IgG-DC) and the control ABPC ADC specific for LRRC15 (e.g., control ABPC LRRC15-IgG-DC) is assessed separately against a panel of LRRC15+ cell lines (e.g., as described herein) and LRRC15- cell lines expressing various densities of antigen, selected using the methods described herein, and optionally against cells expressing transgenic LRRC15, for example, HEK293 cells transfected with LRRC15 using methods known in the art (e.g., Expi293™ Expression System Kit ThermoFisher catalog number: A14635). For validation purposes, prior to use, all cell lines were tested for LRRC15 expression using methods known in the art, such as qPCR, flow cytometry, mRNA RPKM, and antibody staining using art-known anti-LRRC15 antibodies (e.g., as described herein), followed by visualization of the staining using fluorescence microscopy, immunohistochemistry, flow cytometry, ELISA, or other methods known in the art. To assess compound cytotoxicity, cells were seeded at approximately 10-40,000 cells per well in 150 microliters of culture medium and then treated in quadruplicate with graded doses of compound ranging from 1 pM to 1 μM at the beginning of the assay. Cytotoxicity assays were performed for 96 hours after addition of test compounds. Fifty microliters of resazurin dye was added to each well during the final 4-6 hours of incubation, and viable cells were assessed at the end of the culture. Dye reduction was determined by fluorescence spectroscopy using excitation and emission wavelengths of 535 nm and 590 nm, respectively. For analysis, the degree of resazurin reduction by treated cells is compared to untreated control cells to determine percent cytotoxicity. Alternatively, use a WST-8 kit to measure cytotoxicity according to the manufacturer's instructions (e.g., Dojindo Molecular Technologies catalog no. CCK-8).The IC50 is the concentration at which half-maximal killing is observed and is calculated using curve-fitting methods known in the art. Upon analysis of the data, it can be determined that the pH-engineered ABPC ADC specific for LRRC15 and the control ABPC ADC are substantially cytotoxic to one or more LRRC15+ cell lines, but are less toxic to LRRC15- cells. It can also be determined that the pH-engineered ADC specific for LRRC15 is more cytotoxic to one or more LRRC15+ cell lines than the control ABPC ADC specific for LRRC15 because a) they exhibit a greater depth of killing at one or more concentrations, or b) they exhibit a lower IC50, or c) they exhibit a greater ratio of their dissociation constant KD in cells at neutral pH (as described herein) divided by their IC50 in those same cells.

[0549] Additionally, the cytotoxic activity of ABPC specific for LRRC15 can be measured in secondary ADC assays, which are known in the art (e.g., Moradec Catalog No. αHFc-NC-MMAF and Catalog No. αHFc-CL-MMAE, and associated manufacturer's instructions). Briefly, the assay is performed as in the previous paragraph, except that the ABPC specific for LRRC15 is substituted for an ADC specific for LRRC15, and to assess compound cytotoxicity, cells are seeded at approximately 10-40,000 per well in 150 microliters of culture medium and then treated in quadruplicate at the start of the assay with graded doses of 1 pM to 1 μM ABPC specific for LRRC15 (final concentrations in culture medium, premixed with a secondary ADC reagent, Moradec catalog number αHFc-NC-MMAF, final concentration in culture medium of 100 nM and preincubated at 37° C. for 30 minutes prior to addition of the mixture to the culture medium).

[0550] The cytotoxic activity of the pH-engineered ADC specific for LRRC15 and the control ABPC ADC specific for LRRC15 conjugate, as well as the ABPC specific for LRRC15 in secondary ADC assays, is additionally measured by cell proliferation assay using the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al., Cancer Res. 62:5485-5488, 2002):

[0551] 1. A 100 microliter aliquot of cell culture containing approximately 10 4 cells (e.g., LRRC15+ cells described herein) in medium is deposited into each well of a 96-well, opaque-walled plate.

[0552] 2. Control wells are prepared containing medium and no cells.

[0553] 3. ADC specific for LRRC15 is added to experimental wells at concentrations ranging from 1 pM to 1 uM and incubated for 1 to 5 days. Alternatively, in secondary ADC assays, 100 nM secondary ADC reagent (final concentration in culture medium, Moradec catalog number αHFc-NC-MMAF) and ABPC specific for LRRC15 at concentrations ranging from 1 pM to 1 uM (final concentration in culture medium) are premixed and preincubated for 30 minutes at 37°C before adding the mixture to the culture medium and incubated for 1 to 5 days.

[0554] 4. Allow the plate to equilibrate to room temperature for approximately 30 minutes.

[0555] 5. Add a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well.

[0556] 6. Mix contents on an orbital shaker for 2 minutes to induce cell lysis.

[0557] 7. Incubate the plate at room temperature for 10 minutes to allow the luminescent signal to stabilize.

[0558] 8. Luminescence is recorded as RLU = Relative Luminescence Units and reported graphically.

[0559] Example 5. Demonstration of enhanced toxin release of pH-engineered ABPC ADC specific for LRRC15 in LRRC15+ cells compared to control ABPC ADC specific for LRRC15 pH-engineered ADCs specific for LRRC15 (e.g., pH-engineered LRRC15-IgG-DCs) may also demonstrate increased toxin release in LRRC15+ cells compared to control ABPC ADCs specific for LRRC15 (e.g., control ABPC LRRC15-IgG-DCs). Following treatment of LRRC15+ cells with pH-engineered ABPC ADCs specific for LRRC15 and control ABPC ADCs as described herein, an LC-MS / MS method is used to quantify unconjugated (i.e., free) MMAE in the treated LRRC15+ cells (Singh, AP, and Shah, DK. Drug Metabolism and Disposition 45.11(2017):1120-1132). An LC-MS / MS system with an electrospray interface and a triple quadrupole mass spectrometer is used. For MMAE detection, an XBridge BEH amide column (Waters, Milford, MA) was used with mobile phase A as water (with 5 mM ammonium formate and 0.1% formic acid) and mobile phase B as 95:5 acetonitrile / water (with 0.1% formic acid and 1 mM ammonium formate), using a gradient at a flow rate of 0.25 mL / min at 40 °C. The total duration of the chromatographic run was 12 min, and two MRM scans (718.5 / 686.5 and 718.5 / 152.1 amu) were monitored. Deuterated (d8) MMAE (MCE MedChem Express, Monmouth Junction, NJ) was used as the internal standard. An equation for quantifying unconjugated MMAE in biological samples was derived by first dividing the peak area for each drug standard by the peak area obtained for the internal standard. The resulting peak area ratios were then plotted as a function of standard concentration, and the data points were fitted to a curve using linear regression. Three QC samples are included in the lower, middle, and upper ranges of the standard curve to assess the predictive ability of the generated standard curve. The resulting standard curve is then used to estimate the concentrations of MMAE observed in biological samples.For MMAE concentration measurement, treated cell samples were precipitated and reconstituted in fresh medium to a final concentration of 250,000 cells / 100 μL. After spiking the sample with d8-MMAE (1 ng / mL), cell lysis was performed by adding two volumes of ice-cold methanol, followed by a 45-minute freeze-thaw cycle at -20°C. The final cell lysate was obtained by centrifuging the sample at 13,000 rpm for 15 minutes at 4°C, followed by collection of the supernatant. For the preparation of standards and QC samples, fresh cell suspensions (250,000 / 100 μL) were spiked with known concentrations of MMAE and internal standard (d8-MMAE) following the same procedure as for the cell lysate described above. The resulting cell lysate was evaporated and reconstituted in mobile phase B before injection into LC-MS / MS. The concentration of unconjugated MMAE in the lysates of LRRC15+ cells treated with the pH-engineered ADC specific for LRRC15 is observed to be higher than that in LRRC15+ cells treated with the control ABPC ADC specific for LRRC15.

[0560] For tubulin-inhibiting toxins, toxin release is also assessed by monitoring cell viability and cell cycle progression. Approximately 2.0×10^5 LRRC15+ cells are seeded into 96-well flat-bottom plates and treated with the pH-engineered ABPC ADCs specific for LRRC15 and control ABPC ADCs described herein. After treatment, the cells are transferred to a 96-round-bottom plate, the plate is centrifuged at 400 rcf for 2 minutes, and the supernatant is decanted. The decanted cells are stained with Live / Dead eFluor 660. The cells are then centrifuged and washed with FACS buffer (PBS with 2% FBS), after which cell cycle distribution is analyzed using the BD Cycletest™ Plus DNA Kit (Cat. No. 340242). Briefly, cells are resuspended in 76 μl of Solution A and incubated at room temperature for 10 minutes. 61 μL of Solution B is then added, and the cells are incubated at room temperature for another 10 minutes. Finally, 61 μL of cold solution C is added, and the cells are again incubated for 10 minutes at room temperature. Immediately after the final incubation step, the cells are analyzed by flow cytometry (without washing) at a flow rate of 10 μL / sec. Increased G2 / M arrest can be observed upon exposure to the pH-engineered ADCs specific for LRRC15 compared to the control ABPC ADC specific for LRRC15.

[0561] For DNA-damaging toxins (e.g., pyrrolobenzodiazepines or "PBDs"), DNA damage is assessed by measuring phosphorylated histone H2AX (γH2AX). H2AX is normally phosphorylated in response to double-strand breaks in DNA; however, increased levels of γH2AX can also be observed following treatment with DNA-crosslinking toxins, such as PBDs or cisplatin (Huang, X. et al. 2004, Cytometry Part A 58A, 99-110). LRRC15+ cells are treated with the pH-engineered ABPC ADCs specific for LRRC15 described herein and control ABPC ADCs. After treatment, cells are rinsed with PBS and then fixed in 1% methanol-free formaldehyde (Polysciences, Warrington, PA) in PBS for 15 minutes at 0°C. Cells are resuspended in 70% ethanol for at least 2 hours at -20°C. The cells were then washed twice with PBS and suspended in 0.2% Triton® X-100 (Sigma) in a 1% (w / v) solution of BSA (Sigma) in PBS for 30 minutes to suppress nonspecific Ab binding. The cells were centrifuged again (200 g, 5 minutes), and the cell pellet was suspended in 100 μL of 1% BSA containing a 1:800 diluted anti-histone γH2AX polyclonal Ab (Trevigen, Gaithersburg, MD). The cells were then incubated overnight at 4°C, washed twice with PBS, and resuspended in 100 μL of a 1:30 diluted FITC-conjugated F(ab')2 fragment of swine anti-rabbit immunoglobulin (DAKO, Carpinteria, CA) for 30 minutes at room temperature in the dark. Cells are then counterstained with 5 μg / mL PI (Molecular Probes, Eugene, OR) dissolved in PBS containing 100 μg / mL DNase-free RNase A (Sigma) for 20 minutes at room temperature. Cytofluorescence of the FITC γH2AX signal and PI counterstaining are measured using flow cytometry using methods known in the art.When comparing cells within the same stage of the cell cycle (based on total DNA content), treated LRRC15+ cells can be observed to have increased FITC γH2AX signal compared to untreated LRRC15+ cells (which serve as the baseline). Furthermore, LRRC15+ cells treated with the pH-engineered ADC specific for LRRC15 can be observed to have a greater increase in γH2AX levels above baseline than cells treated with the control ABPC ADC specific for LRRC15. In addition to the γH2AX assay, DNA crosslinking can be more directly assessed using the Comet assay (Chandna, S. (2004) Cytometry 61A, 127-133).

[0562] Also, as disclosed herein, pH-engineered and control ABPCs can be assayed using the methods in this example without direct conjugation by performing a secondary ADC assay instead of using a primary conjugated ADC.

[0563] Example 6. Demonstration of increased or decreased half-life of pH-engineered ABPCs specific for LRRC15 compared to control ABPCs specific for LRRC15 One surprising aspect of the pH-engineered ABPCs specific for LRRC15 described by the present invention is their ability to promote increased dissociation of ABPC from LRRC15 in endosomes or lysosomes, resulting in a decreased or increased serum half-life compared to a control ABPC specific for LRRC15 or an ABPC not specific for LRRC15. To demonstrate these properties, a series of animal studies in mice and / or monkeys are performed using the pH-engineered ABPC specific for LRRC15 and the control ABPC specific for LRRC15 using methods known in the art (e.g., Gupta, P., et al. (2016), mAbs, 8:5, 991-997). Briefly, to conduct the mouse study, a single intravenous bolus (e.g., 5 mg / kg) of either the pH-engineered ABPC specific for LRRC15 or the control ABPC specific for LRRC15 is administered via the tail vein to two groups of NOD SCID mice (e.g., Jackson Labs NOD.CB17-Prkdcscid / J Stock No.: 001303) xenografted with an LRRC15+ cell line (e.g., as described herein). Xenografted mice are prepared by growing 1-5 million LRRC15+ cells in vitro and subcutaneously inoculating them into the right flank of the mice. Tumors are size-matched to 300 mm3. Measurements of tumor length (L) and width (W) are obtained via electronic calipers, and volume is calculated according to the following equation: V = L × W^2 / 2. Blood samples are collected via retro-orbital bleed from each group at each of the following time points: 15 minutes, 30 minutes, 1 hour, 8 hours, 24 hours, and 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, and 28 days. Samples are processed to collect serum, and antibody concentrations are quantified using ELISA or other methods known in the art (e.g., PAC assay or MAC assay; Fischer, S.K. et al. (2012), mAbs, 4:5, 623-631, e.g., utilizing anti-human Fc antibody Jackson ImmunoResearch Labs, catalog number 109-006-006).The antibody concentration of the pH-engineered ABPC specific for LRRC15 and the control ABPC specific for LRRC15 is plotted as a function of time. Upon analysis of the data, it may be observed that the pH-engineered ABPC specific for LRRC15 has a significantly longer or shorter serum half-life than the control ABPC specific for LRRC15. If the pH-engineered ABPC and control ABPC specific for LRRC15 are cross-reactive with the mouse homolog of LRRC15, similar experiments can be repeated in non-xenograft mice.

[0564] Optionally, if the pH-engineered ABPC specific for LRRC15 and the control ABPC are cross-reactive with the cynomolgus monkey homolog of LRRC15, similar experiments can be performed in monkeys (e.g., cynomolgus monkeys). Equal numbers of male and female monkeys (e.g., n = 1-2 each) are administered a bolus of either the pH-engineered ABPC specific for LRRC15 or the control ABPC specific for LRRC15 via saphenous vein injection, e.g., at a dose of 1 mg / kg. Alternatively, several different doses of the LRRC15-binding protein are administered across several groups of monkeys. Blood samples are collected via a peripheral or femoral vein at intervals similar to those described above and analyzed for the presence of either the pH-engineered ABPC specific for LRRC15 or the control ABPC specific for LRRC15 using methods known in the art (e.g., ELISA). Upon analyzing the data, it may be observed that the pH-engineered ABPC specific for LRRC15 has a significantly longer or shorter serum half-life than the control ABPC specific for LRRC15. In some cases, this effect is observed only at a specific dose, while in other cases, it is observed across doses. In certain cases, an increased half-life is desirable, especially when less frequent patient dosing is advantageous.

[0565] The half-lives of pH-engineered ABPC ADCs specific for LRRC15 and control ABPC ADCs can also be assessed using the above methods by substituting pH-engineered ABPC ADCs specif...

Claims

1. An antigen binding protein construct (ABPC) comprising a first antigen binding domain (ABD) capable of specifically binding to LRRC15 or an epitope of LRRC15 displayed on the surface of a target mammalian cell; the first ABD comprises an ABD of 15G7, 24D9, or 29F1, optionally with one or more amino acids of the ABD substituted with histidine, aspartic acid, or glutamic acid; Optionally, the dissociation rate of said first ABD at a pH of about 4.0 to 6.5 is faster than the dissociation rate at a pH of about 7.0 to 8.0, or the KD of said first ABD at a pH of about 4.0 to 6.5 is greater than the KD at a pH of about 7.0 to 8.

0.

2. 2. The ABPC of claim 1, wherein the first ABD comprises one of the heavy chain variable domains (HCVDs) of (a) 15G7; (b) 24D9; and (c) 29F1, each HCVD optionally with one or more amino acids substituted with histidine, and optionally the 29F1 HCVD has one or more amino acids substituted with aspartic acid or glutamic acid; and / or wherein the first ABD comprises one of the light chain variable domains (LCVDs) of (a) 15G7; (b) 24D9; and (c) 29F1, each LCVD optionally with one or more amino acids substituted with histidine, and optionally the 29F1 LCVD has one or more amino acids substituted with aspartic acid or glutamic acid.

3. The ABD is (a) HCVD of 15G7; and / or LCVD of 15G7; (b) HCVD of 24D9; and / or LCVD of 24D9; or (c) HCVD of 29F1; and / or LCVD of 29F1; Optionally, each HCVD and / or LCVD has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; Optionally, the ABD is selected from the group consisting of MYT2737 (HCV=SEQ ID NO:1, LCV=SEQ ID NO:64), MYT3315 (HCV=SEQ ID NO:84, LCV=SEQ ID NO:154), MYT8391 (HCV=SEQ ID NO:430, LCV=SEQ ID NO:455), MYT8415 (HCV=SEQ ID NO:382, LCV=SEQ ID NO:489), MYT8417 (HCV=SEQ ID NO:382, LCV=SEQ ID NO:491), MYT8483 (HCV=SEQ ID NO:516, LCV=SEQ ID NO:522), MYT9776 (HCV=SEQ ID NO:571, LCV=SEQ ID NO:517), MYT8094 (HCV=SEQ ID NO:5 76, LCV SEQ ID NO:581), MYT9521 (HCV SEQ ID NO:576, LCV SEQ ID NO:639), MYT9731 (HCV SEQ ID NO:576, LCV SEQ ID NO:700), MYT8416 (HCV SEQ ID NO:382, LCV SEQ ID NO:490), MYT8500 (HCV SEQ ID NO:570, LCV SEQ ID NO:522), MYT9523 (HCV SEQ ID NO:576, LCV SEQ ID NO:641), MYT4174 (HCV SEQ ID NO:84, LCV SEQ ID NO:177), and MYT9507 (HCV SEQ ID NO:576, LCV SEQ ID NO:625); or Optionally, the ABD is selected from antibodies comprising a pair of heavy and light chain polypeptide sequences that are at least 95% identical to the pairs of sequences set forth in SEQ ID NOs: 724 and 725; 726 and 727; 728 and 729; 730 and 731; 730 and 732; 733 and 734; 735 and 736; 737 and 738; 737 and 739; and 737 and 740. The ABPC of claim 2.

4. The HCVD method includes: (a) HCVD of 15G7 comprising the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382; (b) HCVD of 24D9 comprising the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516; and (c) HCVD of 29F1 comprising the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576; and / or The LCVD method includes: (a) an LCVD of 15G7 comprising the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455; (b) an LCVD of 24D9 comprising the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522; and (c) the LCVD of 29F1 comprising the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581; 2. The ABPC of claim 1, wherein optionally each HCVD and / or LCVD has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid.

5. the first ABD comprises a HCVD comprising CDR1, 2, and 3 of one of (a) SEQ ID NOs: 379-381; (b) SEQ ID NOs: 513-515; and (c) SEQ ID NOs: 573-575, optionally wherein each set of three CDRs collectively has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid; and / or 2. The ABPC of claim 1, wherein the first ABD comprises an LCVD comprising the CDRs 1, 2, and 3 of one of: (a) SEQ ID NOs: 452-454; (b) SEQ ID NOs: 519-521; and (c) SEQ ID NOs: 578-580, and optionally each set of three CDRs collectively has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid.

6. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein said HCVD comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516 selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110; and (c) an HCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCVD comprises histidine, aspartic acid, or glutamic acid at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: D positions selected from 31, 56, and 99; and E positions selected from 59.

7. The first ABD is (a) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein said LCVD comprises a histidine at one or more positions in SEQ ID NO:451 or SEQ ID NO:455 selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97; (b) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522 selected from the group consisting of 35 and 97; and (c) an LCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCVD comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions in SEQ ID NO:577 or SEQ ID NO:581 selected from the group consisting of: D positions selected from 27, 28, 31, 52 and 56; and E positions selected from 51 and 56.

8. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCVD comprises histidine at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at two or more positions in SEQ ID NO:512 or SEQ ID NO:516; and (c) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCVD comprises histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO: 572 or SEQ ID NO: 576; and / or The first ABD is (a) an LCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises histidines at two or more positions in SEQ ID NO:451 or SEQ ID NO:455, including one pair of positions selected from the group consisting of: 30, 32; 30, 92; 30, 93; 30, 96; 32, 92; 32, 93; 32, 96; 92, 93; 92, 96 and 93, 96; (b) an LCVD comprising at least one sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:

522.

6. The ABPC of any one of claims 1 to 5, comprising one of the following LCVDs: (a) an LCVD comprising a sequence that is at least 90% identical to SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCVD comprises a histidine at two or more positions in SEQ ID NO: 518 or SEQ ID NO: 522; and (b) an LCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCVD comprises a histidine, an aspartic acid, or a glutamic acid at two or more positions in SEQ ID NO: 577 or SEQ ID NO:

581.

9. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO: 451 or SEQ ID NO: 455 selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516 selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522 selected from the group consisting of 35 and 97; and (c) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCVD comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of: D positions selected from 31, 56, and 99; and E positions selected from 59 in SEQ ID NO:572 or SEQ ID NO:576; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCVD comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of: D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO:577 or SEQ ID NO:

581.

10. the first ABD comprises a HCVD comprising a sequence set forth in SEQ ID NO:378, SEQ ID NO:382, one of SEQ ID NOs:383-424, one of SEQ ID NOs:425-450, SEQ ID NO:512, SEQ ID NO:516, one of SEQ ID NOs:523-567, one of SEQ ID NOs:568-571, SEQ ID NO:572, SEQ ID NO:576, one of SEQ ID NOs:582-624, or one of SEQ ID NOs:653-695; and / or or wherein the first ABD comprises an LCVD comprising a sequence set forth in SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, SEQ ID NOs:486-511, SEQ ID NO:518, SEQ ID NO:522, SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, or one of SEQ ID NOs:696-723; or 2. The ABPC of claim 1, wherein the first ABD comprises an HCVD and an LCVD that comprise an HCDR and an LCDR, respectively, present in the HCVD and LCVD of one of 15G7, 24D9, and 29F1.

11. The first ABD comprises an HCVD comprising a sequence set forth in SEQ ID NO:378, SEQ ID NO:382, one of SEQ ID NOs:383-424, or one of SEQ ID NOs:425-450, and / or an LCVD comprising a sequence set forth in SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, or one of SEQ ID NOs:486-511, wherein the first ABD comprises (a) an HCVD comprising a sequence set forth in SEQ ID NO:378 or SEQ ID NO:382, and a sequence set forth in SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, 2. The ABPC of claim 1, wherein the ABPC does not comprise an LCVD that does not comprise the sequence set forth in SEQ ID NO: 467, one of SEQ ID NOs: 477-478, or one of SEQ ID NOs: 480-482; or (b) an LCVD of SEQ ID NO: 451 or SEQ ID NO: 455, and an HCVD that does not comprise the sequence set forth in SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 396, SEQ ID NO: 403, one of SEQ ID NOs: 417-419, one of SEQ ID NOs: 423-424, one of SEQ ID NOs: 426-430, one of SEQ ID NOs: 438-440, SEQ ID NO: 444, or SEQ ID NO:

450.

12. 12. The ABPC of any one of claims 1 to 11, wherein the ABPC is degraded in the target mammalian cell after internalization of the ABPC by the target mammalian cell; and optionally, the ABPC further comprises a conjugated toxin, radioisotope, drug, or small molecule.

13. 13. The conjugated ABPC of claim 12, wherein the conjugated ABPC provides an increase in toxin release in the target mammalian cell compared to the same amount of a control conjugated ABPC; optionally, the increased toxin release is at least 20%, 50%, 2-fold, or 5-fold.

14. 14. The ABPC of claim 12 or 13, wherein the ABPC provides an increase in target mammalian cell killing compared to the same amount of a control ABPC; optionally, the increased cell killing is at least 20%, 50%, 2-fold, or 5-fold.

15. 15. The ABPC of any one of claims 1 to 14, wherein the ABPC provides an increase in endolysosomal delivery in the target mammalian cell compared to the same amount of a control ABPC; optionally, the increase in delivery is at least a 20%, 50%, 2-fold, or 5-fold increase.

16. 16. The ABPC of any one of claims 1 to 15, wherein the ABPC results in less or no detectable reduction in the level of LRRC15 displayed on the surface of the target mammalian cells compared to the same amount of a control ABPC.

17. A composition, optionally a pharmaceutical composition, comprising an effective amount of an ABPC according to any one of claims 1 to 16.

18. 1. A composition comprising an effective amount of an antigen binding protein construct (ABPC), the first ABD being capable of specifically binding to LRRC15 or an epitope of LRRC15 present on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug, or small molecule, optionally wherein (a) the dissociation rate of said first ABD at a pH of about 4.0 to 6.5 is faster than the dissociation rate at a pH of about 7.0 to 8.0; or the dissociation constant (KD) of said first ABD at a pH of about 4.0 to 6.5 is faster than the dissociation rate at a pH of about 7.0 to 8.

0. is greater than KD at a pH of about 7.0 to 8.0; and (b) the composition provides one or more of: (i) an increase in toxin release in said target mammalian cells compared to a composition comprising the same amount of a control ABPC; (ii) an increase in target mammalian cell killing compared to a composition comprising the same amount of a control ABPC; and (iii) an increase in endolysosomal delivery in said target mammalian cells compared to a composition comprising the same amount of a control ABPC.

19. 19. The composition of claim 18, wherein the first ABD comprises one of the heavy chain variable domains (HCVDs) of (a) 15G7; (b) 24D9; and (c) 29F1, each HCVD optionally with one or more amino acids substituted with histidine, and optionally the 29F1 HCVD has one or more amino acids substituted with aspartic acid or glutamic acid; and / or the first ABD comprises one of the light chain variable domains (LCVDs) of (a) 15G7; (b) 24D9; and (c) 29F1, each LCVD optionally with one or more amino acids substituted with histidine, and optionally the 29F1 LCVD has one or more amino acids substituted with aspartic acid or glutamic acid.

20. The ABD is (a) HCVD of 15G7; and / or LCVD of 15G7; (b) HCVD of 24D9; and / or LCVD of 24D9; or (c) HCVD of 29F1; and / or LCVD of 29F1; Optionally, each HCVD and / or LCVD has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid; Optionally, the ABD is selected from the group consisting of MYT2737 (HCV=SEQ ID NO:1, LCV=SEQ ID NO:64), MYT3315 (HCV=SEQ ID NO:84, LCV=SEQ ID NO:154), MYT8391 (HCV=SEQ ID NO:430, LCV=SEQ ID NO:455), MYT8415 (HCV=SEQ ID NO:382, LCV=SEQ ID NO:489), MYT8417 (HCV=SEQ ID NO:382, LCV=SEQ ID NO:491), MYT8483 (HCV=SEQ ID NO:516, LCV=SEQ ID NO:522), MYT9776 (HCV=SEQ ID NO:571, LCV=SEQ ID NO:517), MYT8094 (HCV=SEQ ID NO:5 76, LCV SEQ ID NO:581), MYT9521 (HCV SEQ ID NO:576, LCV SEQ ID NO:639), MYT9731 (HCV SEQ ID NO:576, LCV SEQ ID NO:700), MYT8416 (HCV SEQ ID NO:382, LCV SEQ ID NO:490), MYT8500 (HCV SEQ ID NO:570, LCV SEQ ID NO:522), MYT9523 (HCV SEQ ID NO:576, LCV SEQ ID NO:641), MYT4174 (HCV SEQ ID NO:84, LCV SEQ ID NO:177), and MYT9507 (HCV SEQ ID NO:576, LCV SEQ ID NO:625); or 20. The composition of claim 19, wherein the ABD is selected from antibodies comprising a pair of heavy and light chain polypeptide sequences that are at least 95% identical to the pairs of sequences set forth in SEQ ID NOs: 724 and 725; 726 and 727; 728 and 729; 730 and 731; 730 and 732; 733 and 734; 735 and 736; 737 and 738; 737 and 739; and 737 and 740.

21. The HCVD method includes: (a) HCVD of 15G7 comprising the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382; (b) HCVD of 24D9 comprising the sequence set forth in SEQ ID NO: 512 or SEQ ID NO: 516; and (c) HCVD of 29F1 comprising the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576; and / or The LCVD method includes: (a) an LCVD of 15G7 comprising the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455; (b) an LCVD of 24D9 comprising the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522; and (c) the LCVD of 29F1 comprising the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581; 20. The composition of claim 18, wherein optionally each HCVD and / or LCVD has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid.

22. the first ABD comprises a HCVD comprising CDR1, 2, and 3 of one of (a) SEQ ID NOs: 379-381; (b) SEQ ID NOs: 513-515; and (c) SEQ ID NOs: 573-575, optionally wherein each set of three CDRs collectively has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid; and / or 19. The composition of claim 18, wherein the first ABD comprises an LCVD comprising CDR1, 2, and 3 of one of: (a) SEQ ID NOs: 452-454; (b) SEQ ID NOs: 519-521; and (c) SEQ ID NOs: 578-580, wherein optionally each set of three CDRs collectively has one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid.

23. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein said HCVD comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516 selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110; and (c) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCVD comprises histidine, aspartic acid, or glutamic acid at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: D positions selected from 31, 56, and 99; and E positions selected from 59.

24. The first ABD is (a) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein said LCVD comprises a histidine at one or more positions in SEQ ID NO:451 or SEQ ID NO:455 selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97; (b) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO: 518 or SEQ ID NO: 522 selected from the group consisting of 35 and 97; and (c) an LCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCVD comprises aspartic acid (D) or glutamic acid (E) at one or more positions in SEQ ID NO: 577 or SEQ ID NO: 581 selected from the group consisting of D positions selected from 27, 28, 31, 52 and 56; and E positions selected from 51 and 56.

25. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCVD comprises histidine at two or more positions in SEQ ID NO: 378 or SEQ ID NO: 382, ​​including one pair selected from the group consisting of: 34, 53; 34, 104; 34, 105; 34, 106; 53, 104; 53, 105 and 53, 106; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at two or more positions in SEQ ID NO:512 or SEQ ID NO:516; and (c) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCVD comprises histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO: 572 or SEQ ID NO: 576; and / or The first ABD is (a) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises histidines at two or more positions in SEQ ID NO:451 or SEQ ID NO:455, including one pair of positions selected from the group consisting of: 30, 32; 30, 92; 30, 93; 30, 96; 32, 92; 32, 93; 32, 96; 92, 93; 92, 96 and 93, 96; (b) an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 518 or SEQ ID NO: 522, wherein the LCVD comprises a histidine at two or more positions in SEQ ID NO: 518 or SEQ ID NO: 522; and (c) an LCVD comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCVD comprises histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO: 577 or SEQ ID NO:

581.

26. The first ABD is (a) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382 selected from the group consisting of 32, 34, 53, 60, 103, 104, 105, 109, and 110; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO: 451 or SEQ ID NO: 455 selected from the group consisting of 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97; (b) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises a histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516 selected from the group consisting of 30, 31, 32, 52, 53, 58, 59, 60, 98, 105, 106, and 110; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD comprises a histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522 selected from the group consisting of 35 and 97; and 23. The composition of claim 18, wherein the composition comprises one of: (c) an HCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCVD comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of: D positions selected from 31, 56, and 99; and E positions selected from 59 in SEQ ID NO: 572 or SEQ ID NO: 576; and / or an LCVD comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 581, wherein the LCVD comprises an aspartic acid (D) or a glutamic acid (E) at one or more positions selected from the group consisting of: D positions selected from 27, 28, 31, 52, and 56; and E positions selected from 51 and 56 in SEQ ID NO: 577 or SEQ ID NO:

581.

27. the first ABD comprises a HCVD comprising a sequence set forth in SEQ ID NO:378, SEQ ID NO:382, one of SEQ ID NOs:383-424, one of SEQ ID NOs:425-450, SEQ ID NO:512, SEQ ID NO:516, one of SEQ ID NOs:523-567, one of SEQ ID NOs:568-571, SEQ ID NO:572, SEQ ID NO:576, one of SEQ ID NOs:582-624, or one of SEQ ID NOs:653-695; and / or or wherein the first ABD comprises an LCVD comprising a sequence set forth in SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, SEQ ID NOs:486-511, SEQ ID NO:518, SEQ ID NO:522, SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, or one of SEQ ID NOs:696-723; or 20. The composition of claim 18, wherein the first ABD comprises an HCVD and an LCVD that comprise an HCDR and an LCDR, respectively, present in the HCVD and LCVD of one of 15G7, 24D9, and 29F1.

28. the first ABD comprises a HCVD comprising a sequence as set forth in SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, or one of SEQ ID NOs: 425-450, and / or a LCVD comprising a sequence as set forth in SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, or one of SEQ ID NOs: 486-511; 19. The composition of claim 18, wherein the first ABD does not include (a) an HCVD comprising the sequence set forth in SEQ ID NO:378 or SEQ ID NO:382 and an LCVD not comprising the sequence set forth in SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:467, one of SEQ ID NOs:477-478, or one of SEQ ID NOs:480-482; or (b) an LCVD of SEQ ID NO:451 or SEQ ID NO:455 and an HCVD not comprising the sequence set forth in SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:396, SEQ ID NO:403, one of SEQ ID NOs:417-419, one of SEQ ID NOs:423-424, one of SEQ ID NOs:426-430, one of SEQ ID NOs:438-440, SEQ ID NO:444, or SEQ ID NO:

450.

29. 29. The composition of any one of claims 17 to 28, wherein the ABPC is degraded in the target mammalian cell after internalization of the ABPC by the target mammalian cell; and optionally, the ABPC further comprises a conjugated toxin, radioisotope, drug, or small molecule.

30. 30. The composition of claim 29, wherein the conjugated ABPC provides an increase in toxin release in the target mammalian cells compared to the same amount of a control conjugated ABPC; optionally, the increased toxin release is at least 20%, 50%, 2-fold, or 5-fold.

31. 31. The composition of claim 29 or 30, wherein the ABPC provides an increase in target mammalian cell killing compared to the same amount of a control ABPC; optionally, the increased cell killing is at least 20%, 50%, 2-fold, or 5-fold.

32. 32. The composition of any one of claims 17-31, wherein the composition provides an increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC; optionally, the increase in delivery is at least a 20%, 50%, 2-fold, or 5-fold increase.

33. 33. The composition of any one of claims 17 to 32, wherein the ABPC results in less or no detectable reduction in the level of LRRC15 displayed on the surface of the target mammalian cells compared to a composition comprising the same amount of a control ABPC.

34. the target mammalian cells are cancer cells or cancer-associated fibroblasts (CAFs); optionally, the cancer cells are part of or from a sarcoma optionally selected from leiomyosarcoma, osteosarcoma, and chondrosarcoma; and / or The ABPC of any one of claims 1 to 16 or the composition of any one of claims 17 to 33, wherein the CAFs are positive for LRRC15 expression.

35. the dissociation rate of the ABD at a pH of about 4.0 to 6.5 is at least 10%, 3 times, or 10 times faster than the dissociation rate of the ABD at a pH of about 7.0 to 8.0; and / or the KD of said ABD at a pH of about 4.0 to 6.5 is at least 10%, 3-fold, or 10-fold greater than the KD of said ABD at a pH of about 7.0 to 8.0; and / or 35. The ABPC or composition of claim 34, wherein the ABPC is cytotoxic or cytostatic to the cancer cells or CAFs.

36. the ABPC is cross-reactive with non-human primate LRRC15 and human LRRC15; the ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, and one or both of rat LRRC15 and mouse LRRC15; the ABPC is cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15; and / or 36. The ABPC or composition of claim 34 or 35, wherein the ABD binds to an epitope of LRRC15 present on the surface of a cell from an Old World monkey.

37. 37. The ABPC or composition of any one of claims 1 to 36, wherein the ABPC comprises a single polypeptide, and optionally the ABD is selected from the group consisting of a VH domain, a VHH domain, a VNAR domain, and an scFv.

38. 37. The ABPC or composition of any one of claims 1 to 36, wherein the ABPC is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, sc diabody, sc diabody-CH3, scFv-CH-CL-scFv, HSAbody, sc diabody-HSA, or tandem-scFv.

39. 37. The ABPC or composition of any one of claims 1 to 36, wherein the ABPC comprises two or more polypeptides.

40. The ABPC may be an antibody, a VHH-scAb, a VHH-Fab, a double scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-hole common light chain, a knobs-in-hole assembly, a 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, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, dock and lock 40. The ABPC of claim 39, wherein the ABPC is selected from the group consisting of: ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAC, and PROTAB.

41. 41. The ABPC of any one of claims 12 to 16 and 38 to 40, or the composition of any one of claims 17 to 33, wherein at least one polypeptide of the ABPC is conjugated to the toxin, radioisotope, drug, or small molecule via a cleavable or non-cleavable linker.

42. 42. The ABPC or composition of any one of claims 1-41, wherein the half-life of the ABPC in vivo is increased compared to the half-life of a control ABPC in vivo, optionally wherein the increase is about 5% to 95%, about 10% to 95%, about 30% to 95%, about 50% to 95%, or about 70% to 95% compared to the half-life of a control ABPC in vivo.

43. 43. The ABPC or composition of any one of claims 1 to 42, wherein the ABPC further comprises a second ABD.

44. 44. A method of treating a cancer characterized by having a population of cancer cells and / or CAFs having a predetermined level of LRRC15 or an epitope of LRRC15 present on their surface, the method comprising administering a therapeutically effective amount of the ABPC or composition of any one of claims 1 to 43 to a subject identified as having a cancer characterized by having said population of cancer cells and / or CAFs, thereby treating said cancer.

45. 44. A method of reducing tumor volume in a subject, wherein the tumor is characterized by having a population of cancer cells and / or CAFs having a predetermined level of LRRC15 or an epitope of LRRC15 present on their surface, comprising administering a therapeutically effective amount of the ABPC or composition of any one of claims 1 to 43 to a subject identified as having a cancer characterized by having said population of cancer cells and / or CAFs, thereby reducing the volume of the tumor.

46. 44. A method of inducing cell death in cancer cells and / or CAFs in a subject, said cancer cells and / or CAFs having a predetermined level of LRRC15 or an epitope of LRRC15 present on their surface, comprising administering a therapeutically effective amount of the ABPC or composition of any one of claims 1 to a subject identified as having a cancer characterized by having a population of cancer cells and / or CAFs, thereby inducing said cell death in said cancer cells and / or CAFs.

47. 44. A method of reducing the risk of developing metastases or reducing the risk of developing additional metastases in a subject having cancer, wherein the cancer is characterized by having a population of cancer cells, CAFs, and / or stromal cells that have a predetermined level of LRRC15 or an epitope of LRRC15 present on their surface, comprising administering a therapeutically effective amount of the ABPC or composition of any one of claims 1 to 43 to a subject identified as having a cancer characterized by having said population of cancer cells, CAFs, and / or stromal cells, thereby reducing said risk.

48. An anti-LRRC15 antibody, (a) a heavy chain variable domain (HCVD) and a light chain variable domain (LCVD) comprising sequences that are 90% identical to and contain the same heavy chain CDRs and light chain CDRs as the HCVD and LCVD sequences set forth in one of the following sequence pairs: SEQ ID NOs: 1 and 64, SEQ ID NOs: 84 and 154, SEQ ID NOs: 430 and 455, SEQ ID NOs: 382 and 489, SEQ ID NOs: 382 and 491, SEQ ID NOs: 516 and 522, SEQ ID NOs: 571 and 517, SEQ ID NOs: 576 and 581, SEQ ID NOs: 576 and 639, SEQ ID NOs: 576 and 700, SEQ ID NOs: 382 and 490, SEQ ID NOs: 570 and 522, SEQ ID NOs: 576 and 641, SEQ ID NOs: 84 and 177, and SEQ ID NOs: 576 and 625; or (b) HCVD and LCVD sequences that are 95% identical to and contain the same heavy and light chain CDRs as the HCVD and LCVD sequences set forth in one of the following sequence pairs: SEQ ID NOs: 1 and 64, SEQ ID NOs: 84 and 154, SEQ ID NOs: 430 and 455, SEQ ID NOs: 382 and 489, SEQ ID NOs: 382 and 491, SEQ ID NOs: 516 and 522, SEQ ID NOs: 571 and 517, SEQ ID NOs: 576 and 581, SEQ ID NOs: 576 and 639, SEQ ID NOs: 576 and 700, SEQ ID NOs: 382 and 490, SEQ ID NOs: 570 and 522, SEQ ID NOs: 576 and 641, SEQ ID NOs: 84 and 177, and SEQ ID NOs: 576 and 625; or (c) one of the following pairs of sequences: SEQ ID NOs: 1 and 64, SEQ ID NOs: 84 and 154, SEQ ID NOs: 430 and 455, SEQ ID NOs: 382 and 489, SEQ ID NOs: 382 and 491, SEQ ID NOs: 516 and 522, SEQ ID NOs: 571 and 517, SEQ ID NOs: 576 and 581, SEQ ID NOs: 576 and 639, SEQ ID NOs: 576 and 700, SEQ ID NOs: 382 and 490, SEQ ID NOs: 570 and 522, SEQ ID NOs: 576 and 641, SEQ ID NOs: 84 and 177, SEQ ID NOs: 5 76 and 625, SEQ ID NOs: 724 and 725, SEQ ID NOs: 726 and 727, SEQ ID NOs: 728 and 729, SEQ ID NOs: 730 and 731, SEQ ID NOs: 730 and 732, SEQ ID NOs: 733 and 734, SEQ ID NOs: 735 and 736, SEQ ID NOs: 737 and 738, SEQ ID NOs: 737 and 739, and SEQ ID NOs: 737 and 740, and optionally wherein position 205 of the light chain sequence of the antibody comprises an amino acid other than cysteine.

49. 49. The anti-LRRC15 antibody of claim 48, comprising heavy and light chain sequences having the sequences set forth in one of the following sequence pairs: SEQ ID NOs: 724 and 725, SEQ ID NOs: 726 and 727, SEQ ID NOs: 728 and 729, SEQ ID NOs: 730 and 731, SEQ ID NOs: 730 and 732, SEQ ID NOs: 733 and 734, SEQ ID NOs: 735 and 736, SEQ ID NOs: 737 and 738, SEQ ID NOs: 737 and 739, and SEQ ID NOs: 737 and 740, wherein optionally position 205 of the light chain sequence of the antibody comprises an amino acid other than cysteine.

50. 50. The anti-LRRC15 antibody of claim 48 or 49 for use in treating a subject having an LRRC15+ tumor comprising LRRC15+ cancer cells and / or LRRC15+ CAFs.

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