LRRC-15 binding protein constructs and uses thereof

Abstract

Provided herein are antigen binding protein constructs (ABPCs), antibodies, and antibody drug conjugates (ADCs) that bind to LRRC15 and uses thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 415,594 filed on October 12, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically as an XML file named "LRRC15_PRO_SL.XML". The size of the XML file, created on September 22, 2023, is 1,035,741 bytes. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of biotechnology, and more particularly to antigen binding molecules. Summary of the invention

[0006] Antibody-drug conjugates (ADCs) have been designed to combat a variety of diseases. A particular advantage of this approach is that ADCs can have cytostatic or cytotoxic effects. Despite years of development, improved ADCs are desirable.

[0007] 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 pathologies including cancer, and increased killing of target mammalian cells.

[0008] Provided herein is a pharmaceutical composition comprising an effective amount of an ABPC, the ABPC comprising: a first antigen binding domain (ABD) capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell, wherein: (a) the dissociation rate of the first ABD at a pH of about 4.0 to about 6.5 is faster than the dissociation rate at a pH of about 7.0 to about 8.0; and / or (b) the dissociation constant (KD) of the first ABD at a pH of about 4.0 to about 6.5 is greater than the KD at a pH of about 7.0 to about 8.0.

[0009] Non-limiting examples of LRRC15-specific ABPCs and antibodies may include those comprising the variable domains and / or CDRs disclosed in WO2022 / 216653A1 (to Mythic Therapeutics and incorporated herein by reference), including those ABPCs, antibodies, variable domains, and / or CDRs present in MYT2737 and MYT3315.

[0010] Encouraged by their initial findings, and reasoning that anti-LRRC15-ADCs may prove useful against both LRRC15+ tumors as well as LRRC15- tumors associated with LRRC15+ stromal cells, Applicants generated the novel anti-LRRC15 antibodies disclosed herein.

[0011] As described in further detail herein, the following are non-limiting examples of pH-dependent anti-LRRC15 ABPCs that exhibit strong target-specific binding at physiological pH, enhanced dissociation at acidic pH, enhanced internalization into LRRC15+ cells, and enhanced cytotoxicity when complexed or conjugated with the following toxic payloads: 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: 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:639) 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).

[0012] In some embodiments, the anti-LRRC15 ABPCs have the same heavy and light chains, heavy and light chain variable domains, and / or heavy and light chain CDRs (e.g., as determined by, e.g., Kabat, Chothia, and / or IMGT numbering schemes) as MYT2737-c (comprising SEQ ID NOs: 724 and 725); MYT3315-c (comprising SEQ ID NOs: 726 and 727); MYT8391-c (comprising SEQ ID NOs: 728 and 729); MYT8415-c (comprising SEQ ID NOs: 730 and 731); MYT8417-c (comprising SEQ ID NOs: 730 and 732); MYT8483-c (comprising SEQ ID NOs: 733 and 734); MYT9776-c (comprising SEQ ID NOs: 735 and 736); MYT9521-c (comprising SEQ ID NOs: 736 and 737); NO: 737 and 738); MYT9731-c (comprising SEQ ID NO: 737 and 739); or MYT8094-c (comprising 737 and 740). Each of the foregoing can be conjugated to toxins at various positions, including position 205 of the corresponding light chain (i.e., the sequence shown in one of SEQ ID NO: 725, 727, 729, 731, 732, 734, 736, 738, 739, and 740). In some embodiments, position 205 of the light chain comprises an amino acid other than cysteine ​​(e.g., valine, leucine, isoleucine, or alanine). In some embodiments, toxin conjugation can be performed at other positions known to those skilled in the art. In still other embodiments, the toxin can be attached by hinge conjugation or any other suitable attachment method.

[0013] In some embodiments, the first ABD (also referred to as a "LRRC15 binding domain") comprises a heavy chain variable (HCV) domain of one of the following: (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. The first ABD may also include an 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 include 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 any corresponding humanized counterpart thereof. When preceded by "hu" (e.g., "hu15G7"), it means a humanized clone.

[0014] In some embodiments, the anti-LRRC15 antibody comprises a heavy chain and a light chain having a polypeptide sequence as shown in SEQ ID NO: 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; or 737 and 740. In other embodiments, the anti-LRRC15 antibody comprises a heavy chain and a light chain having a polypeptide sequence as shown in SEQ ID NO: 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; or 737 and 740, wherein position 205 of the corresponding light chain is not cysteine. In some embodiments, the anti-LRRC15 antibody comprises a light chain having valine, leucine, or isoleucine at position 205.

[0015] In some embodiments, the anti-LRRC15 antibody may comprise a heavy chain and a light chain comprising a favorable "developability mutation", e.g., as described in Table 33. As used herein, "developability mutation" means a change in the amino acid sequence of an ABPC (including an antibody) that tends to improve certain biophysical properties (e.g., reduce the likelihood of aggregation). In other embodiments, the heavy chain and the light chain may comprise conservative amino acid substitutions relative to favorable developability mutations.

[0016] In some embodiments, the composition comprising the ABPC or antibody provides one or more of the following: increased toxin release in target mammalian cells compared to a composition comprising the same amount of a control antibody; increased killing of target mammalian cells compared to a composition comprising the same amount of a control antibody; and / or increased endolysosomal delivery in the target mammalian cells compared to a composition comprising the same amount of a control antibody.

[0017] In some embodiments, the composition comprising the antibody results in reduced or undetectable reduction in the level of LRRC15 presented on the surface of a target mammalian cell compared to a composition comprising the same amount of a control antibody.

[0018] In some embodiments, after the antibody is internalized by the target mammalian cell, the antibody is degraded in 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 causes an increased selectivity for cancer cells relative to non-cancerous cells.

[0019] 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.

[0020] In some embodiments, the in vivo half-life of the antibody is increased compared to the in vivo half-life of a control antibody.

[0021] In some embodiments, the first ABD comprises a light chain variable (LCV) domain of one of the following: (a) an LCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) an LCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) an LCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid or glutamic acid. The first ABD may also comprise an LCV domain of MYT2737 or MYT3315.

[0022] In some embodiments, the first ABD comprises one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; and / or the LCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and / or the LCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid or glutamic acid; and / or the LCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid or glutamic acid. In the case where the wild-type amino acid is already histidine, alanine can be substituted instead.

[0023] In some embodiments, the HCV domain includes one of the following: (a) an HCV domain of 15G7, which includes SEQ ID NO:378 or a humanized version thereof (e.g., SEQ ID NO:382); (b) an HCV domain of 24D9, which includes SEQ ID NO:512 or a humanized version thereof (e.g., SEQ ID NO:516); and (c) an HCV domain of 29F1, which includes SEQ ID NO:572 or a humanized version thereof (e.g., SEQ ID NO:576). In some embodiments, the LCV domain comprises one of the following: (a) an LCV domain of 15G7 comprising SEQ ID NO:451 or a humanized version thereof (e.g., SEQ ID NO:455); (b) an LCV domain of 24D9 comprising SEQ ID NO:518 or a humanized version thereof (e.g., SEQID NO:522); and (c) an LCV domain of 29F1 comprising SEQ ID NO:577 or a humanized version thereof (e.g., SEQID NO:581).

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

[0025] In some embodiments, the first LRRC15 binding domain comprises an LCV domain of one of the following: (a) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 519-521 are substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 578-580, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0026] In some embodiments, the first LRRC15 binding domain comprises one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: A total of one or more amino acid positions in NO:519-521 are substituted with histidine; and (c) an HCV domain, the HCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:573-575, respectively, optionally a total of one or more amino acid positions in SEQID NO:573-575 are substituted with histidine, aspartic acid or glutamic acid; and / or an LCV domain, the LCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:578-580, respectively, optionally a total of one or more amino acid positions in SEQID NO:578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0027] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises histidine (or alanine, where 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 shown in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises histidine (or alanine, where histidine is already present) at one or more positions in SEQ ID NO: 378 or SEQ ID NO: 382; NO:516 includes 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; and (c) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain includes 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 an LCV domain of one of the following: (a) an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:582. NO:577 or SEQ ID NO:581 comprises histidine, aspartic acid (D) or glutamic acid (E) at one or more positions selected from the group consisting of: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

[0028] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:378 or SEQ ID NO:382, wherein the HCV domain comprises histidine at two or more positions in SEQ ID NO:378 or SEQ ID NO:382, including a pair of positions selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516. In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain, which is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises histidine at two or more positions in SEQ ID NO:451 or SEQ ID NO:455 comprising a 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, which is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522.

[0029] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises 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 shown in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises 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; and (b) an HCV domain that is identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: NO:516, wherein the HCV domain comprises 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 an LCV domain, the LCV domain being at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 HCV domain, the HCV domain being at least 90% identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises histidine at one or more positions selected from the group consisting of: 35 and 97 in SEQ ID NO:572 or SEQ ID NO:576. NO:576 comprises aspartic acid (D) or glutamic acid (E) at one or more positions selected from the group consisting of: a D position selected from 31, 56 and 99; or an E position 59; and / or a LCV domain, wherein the LCV domain is at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCV domain comprises aspartic acid (D) or glutamic acid (E) at one or more positions selected from the group consisting of: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

[0030] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 512, SEQ ID NO: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: NO:653-695; and / or the first ABD includes an LCV domain of one of the following: (a) an LCV domain, the LCV domain having a sequence of one of the following: SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, and one of SEQ ID NOs:486-511; (b) an LCV domain, the LCV domain comprising a sequence of SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H substitution) or SEQ ID NO:518 (with G97H substitution); and (c) an LCV domain, the LCV domain comprising a sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and one of SEQ ID NOs:696-723.

[0031] In some embodiments, the first ABD includes one of the following: (a) an HCV domain comprising a sequence of one of SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain having a sequence of one of SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, wherein the first ABD does not include (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382 and an LCV domain comprising a sequence that is not one of 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, and SEQ ID NO: NO:480-482; or (ii) an LCV domain comprising a sequence of SEQ ID NO:451 or SEQ ID NO:455 and an HCV domain comprising a sequence that is not one of: 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, and SEQ ID NO:450; (b) an HCV domain comprising a sequence of one of: SEQ ID NO:512, SEQ ID NO:516, one of SEQ ID NOs:523-567, and one of SEQ ID NOs:568-571; and / or an LCV domain comprising a sequence of: SEQ ID NO:514, SEQ ID NO:516, one of SEQ ID NOs:523-567, and one of SEQ ID NOs:568-571; NO:518, SEQ ID NO:522, SEQ ID:518 (with an L35H substitution) or SEQ ID NO:518 (with a G97H substitution), wherein the first ABD does not include (i) an LCV domain comprising a sequence of SEQ ID NO:512 or SEQ ID NO:516 and a sequence that is not one of SEQ ID NO:518 (with an L35H substitution) and SEQ ID NO:518 (with a G97H substitution);Or (ii) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID: 518 (with L35H) or SEQ ID NO: 518 (with G97H) and an HCV domain comprising a sequence that does not include one of the following: one of SEQ ID NOs: 527-529, one of SEQ ID NOs: 535-536, one of SEQ ID NOs: 541-543, SEQ ID NO: 552, one of SEQ ID NOs: 559-560, and SEQ ID NO: 564; and (c) 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 one of the following: SEQ ID NO: 577, SEQ ID NO: 581, one of SEQ ID NOs: 625-652, and SEQ ID NO: NO:696-723, wherein the first ABD does not include (i) an HCV domain comprising a sequence of SEQ ID NO:572 or SEQ ID NO:576 and an LCV domain comprising a sequence that is not one of the following: SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:632, SEQ ID NO:638, and SEQ ID NO:642; or (ii) an LCV domain comprising a sequence of SEQ ID NO:577 or SEQ ID NO:581 and an HCV domain comprising a sequence that is not one of the following: SEQ ID NO:587, SEQ ID NO:598, SEQ ID NO:611, and SEQ ID NO:672. ;

[0032] In some embodiments, after the ABPC is internalized by the target mammalian cell, the ABPC is degraded in the target mammalian cell. In some embodiments, the ABPC comprises a conjugated toxin, radioisotope, drug or small molecule.

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

[0034] In some embodiments, the composition provides an increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 20% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 50% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 2-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 5-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0035] In some embodiments, the composition provides an increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 20% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 50% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 2-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 5-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs.

[0036] In some embodiments, the composition causes less reduction in the level of LRRC15 presented on the surface of the target mammalian cell than a composition comprising the same amount of a control ABPC. In some embodiments, the composition does not cause a detectable reduction in the level of LRRC15 presented on the surface of the target mammalian cell.

[0037] Also provided herein is a pharmaceutical composition comprising an effective amount of an antigen binding protein construct (ABPC), the ABPC comprising: a first ABD, the first ABD being capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell; and a conjugated toxin, a radioisotope, a drug or a 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 greater than the dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5. D ) is greater than K at a pH of about 7.0 to about 8.0 D ; and (b) the composition provides one or more of the following: increased toxin release in the target mammalian cells compared to a composition comprising the same amount of a control ABPC; increased target mammalian cell killing compared to a composition comprising the same amount of a control ABPC; and increased endolysosomal delivery in the target mammalian cells compared to a composition comprising the same amount of a control ABPC.

[0038] In some embodiments, the first ABD comprises the HCV domain of one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises the LCV domain of one of the following: (a) the LCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the LCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the LCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0039] In some embodiments, the first ABD comprises one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate; and / or the LCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate.

[0040] In some embodiments, the HCV domain comprises one of the following: (a) a HCV domain of 15G7 comprising SEQ ID NO: 378 or a humanized version thereof (e.g., SEQ ID NO: 382); (b) a HCV domain of 24D9 comprising SEQ ID NO: 512 or a humanized version thereof (e.g., SEQ ID NO: 516); and (c) a 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 the following: (a) a LCV domain of 15G7 comprising SEQ ID NO: 451 or a humanized version thereof (e.g., SEQ ID NO: 455); (b) a LCV domain of 24D9 comprising SEQ ID NO: 518 or a humanized version thereof (e.g., SEQ ID NO: 522); and (c) a LCV domain of 29F1 comprising SEQ ID NO: NO:577 or a humanized version thereof (e.g., SEQ ID NO:581).

[0041] In some embodiments, the first ABD comprises a HCV domain, which comprises one of the following: (a) a HCV domain, which comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, and optionally one or more amino acid positions in total in SEQ ID NOs: 379-381 are substituted by histidine; (b) a HCV domain, which comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, and optionally one or more amino acid positions in total in SEQ ID NOs: 513-515 are substituted by histidine; and (c) a HCV domain, which comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 573-575, respectively, and optionally one or more amino acid positions in total in SEQ ID NOs: 573-575 are substituted by histidine, aspartic acid or glutamic acid.

[0042] In some embodiments, the first LRRC15 binding domain comprises an LCV domain of one of the following: (a) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 519-521 are substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 578-580, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0043] In some embodiments, the first LRRC15 binding domain comprises one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: A total of one or more amino acid positions in NO:519-521 are substituted with histidine; and (c) an HCV domain, the HCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:573-575, respectively, optionally a total of one or more amino acid positions in SEQID NO:573-575 are substituted with histidine, aspartic acid or glutamic acid; and / or an LCV domain, the LCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:578-580, respectively, optionally a total of one or more amino acid positions in SEQID NO:578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0044] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or SEQ ID NO: 382, ​​wherein the HCV domain comprises 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 HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises 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 HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: 516 The sequence shown in SEQ ID NO:572 or SEQ ID NO:576 is at least 90% identical, wherein the HCV domain comprises histidine, aspartic acid or glutamic acid at one or more positions selected from position 31, position 56, position 59 and position 99 in SEQ ID NO:572 or SEQ ID NO:576.

[0045] In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:582. NO:577 or SEQ ID NO:581 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.

[0046] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain comprises histidine at two or more positions in SEQ ID NO:378 or SEQ ID NO:382 comprising a pair selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516. In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain, which is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises histidine at two or more positions in SEQ ID NO:451 or SEQ ID NO:455 comprising a 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, which is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522.

[0047] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises 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 shown in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises 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 HCV domain that is identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: NO:516, wherein the HCV domain comprises 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 an LCV domain, the LCV domain being at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 HCV domain, the HCV domain being at least 90% identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises histidine at one or more positions selected from the group consisting of: 35 and 97 in SEQ ID NO:572 or SEQ ID NO:576. NO:576 comprises histidine, aspartic acid or glutamic acid at one or more positions selected from the group consisting of: 31, 56, 59 and 99; and / or a LCV domain, wherein the LCV domain is at least 90% identical to the sequence shown 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.

[0048] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 512, SEQ ID NO: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695.

[0049] In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain having a sequence of one of the following: SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, and one of SEQ ID NOs:486-511; (b) an LCV domain comprising a sequence of one of the following: SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H), or SEQ ID NO:518 (with G97H); and (c) an LCV domain comprising a sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and one of SEQ ID NOs:696-723.

[0050] In some embodiments, the first ABD comprises one of: (a) an HCV domain comprising a sequence of any one of SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain, the LCV domain being one of SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, wherein the first ABD does not include (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382 and an LCV domain comprising a sequence that is not one of 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, and SEQ ID NO: 479. NO:480-482; and (ii) an LCV domain comprising a sequence of SEQ ID NO:451 or SEQ ID NO:455 and an HCV domain comprising a sequence that does not include one of the following: one of SEQ ID NO:527-529, one of SEQ ID NO:535-536, one of SEQ ID NO:541-543, one of SEQ ID NO:552, one of SEQ ID NO:559-560, and SEQ ID NO:564; (b) an HCV domain comprising a sequence of any one of the following: SEQ ID NO:512, 516, 523-567, or 568-571; and / or an LCV domain comprising a sequence of SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H), or SEQ ID NO:518 (with G97H), wherein the first ABD does not include (i) a sequence comprising SEQ ID NO:512 or SEQ ID NO:516 and an LCV domain comprising a sequence that is not one of SEQ ID NO:518 (with L35H substitution) and SEQ ID NO:518 (with G97H substitution);and (ii) an LCV domain comprising the sequence of SEQ ID NO: 518, SEQ ID NO: 522, SEQ ID: 518 (with L35H) or SEQ ID NO: 518 (with G97H) and an HCV domain comprising a sequence that does not include one of the following: one of SEQ ID NOs: 527-529, one of SEQ ID NOs: 535-536, one of SEQ ID NOs: 541-543, SEQ ID NO: 552, one of SEQ ID NOs: 559-560, and SEQ ID NO: 564; and (c) an HCV domain comprising the sequence of any one of the following: SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695; and / or an LCV domain, the LCV domain being one of the following: SEQ ID NO:577, 581, 625-652 or 696-723, wherein the first ABD does not include (i) an HCV domain comprising a sequence of SEQ ID NO:572 or SEQ ID NO:576 and an LCV domain comprising a sequence that is not one of the following: SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:632, SEQ ID NO:638 and SEQ ID NO:642; and (ii) an LCV domain comprising a sequence of SEQ ID NO:577 or SEQ ID NO:581 and an HCV domain comprising a sequence that is not one of the following: SEQ ID NO:587, SEQ ID NO:598, SEQ ID NO:611 and SEQ ID NO:672. ;

[0051] In some embodiments, the composition provides an increase in toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 20% increase in toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 50% increase in toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 2-fold increase in toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 5-fold increase in toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0052] In some embodiments, the composition provides an increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 20% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 50% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 2-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition provides at least a 5-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, 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.

[0053] In some embodiments, the composition provides at least a 20% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 50% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 2-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs. In some embodiments, the composition provides at least a 5-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of control ABPCs.

[0054] In some embodiments, the composition causes less reduction in the level of LRRC15 presented on the surface of the target mammalian cell than a composition comprising the same amount of a control ABPC. In some embodiments, the composition does not cause a detectable reduction in the level of LRRC15 presented on the surface of the target mammalian cell. In some embodiments, the target mammalian cell is a cancer cell.

[0055] In some embodiments, the ABD has an off-rate at a pH of about 4.0 to about 6.5 that is at least 10% faster than the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the off-rate of the ABD at a pH of about 4.0 to about 6.5 is at least 3 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the off-rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the ABD has an off-rate at a pH of about 4.0 to about 6.5 that is at least 10 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. D than the K of the ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the ABD has a K of about 10% greater than the ABD at a pH of about 4.0 to about 6.5. D is the K of the ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the ABD has a K of at least 3 times greater than that of the ABD at a pH of about 4.0 to about 6.5. D is the K of the ABD at a pH of about 7.0 to about 8.0 D At least 10 times.

[0056] In some embodiments, the ABPC is cytotoxic or cytostatic to the target mammalian cells. 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 a cell from an Old World Monkey.

[0057] 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 a scFv. In some embodiments, the ABPC is a BiTe, (scFv) 2 , nanobody, nanobody-HSA, DART, TandAb, sc bifunctional antibody, sc bifunctional antibody-CH3, scFv-CH-CL-scFv, HSAbody, sc bifunctional antibody-HSA or tandem scFv.

[0058] In some embodiments, the ABPC comprises two or more polypeptides. In some embodiments, the ABPC is selected from the group consisting of antibodies, VHH-scAb, VHH-Fab, double scFab, F(ab') 2 , bifunctional antibody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common light chain of knob and hole, knob and 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, bifunctional antibody-CH3, trifunctional antibody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab') 2 -scFv 2 , scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, docking lock antibody, ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAB and PROTAC.

[0059] In some embodiments, the ABPC or the ABD comprises a proteolytic targeting antibody (PROTAB) (as described, for example, in Marei et al., "Antibody targeting of E3ubiquitin ligases for receptor degradation". Nature, October 6, 2022). In some embodiments, PROTAB or PROTAC can link a cell surface E3 ubiquitin ligase to a transmembrane protein, causing target degradation both in vitro and in vivo.

[0060] In some embodiments, at least one polypeptide of the ABPC is conjugated to the toxin, the radioisotope, the drug, or the small molecule via a cleavable linker. In some embodiments, at least one polypeptide of the ABPC is conjugated to the toxin, the radioisotope, the drug, or the small molecule via a non-cleavable linker.

[0061] In some embodiments, the in vivo half-life of the ABPC is increased compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 5% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 10% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 30% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 50% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 70% to about 95% compared to the in vivo half-life of the control ABPC. In other embodiments, the in vivo half-life can be reduced.

[0062] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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 3 times 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 D ) is greater than K at a pH of about 7.0 to about 8.0 D No more than 3 times larger.

[0063] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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 2 times 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 D ) exceeds the K at a pH of about 7.0 to about 8.0 D No more than 2 times larger.

[0064] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is D ) is greater than K at a pH of about 7.0 to about 8.0 D No more than 1 times larger.

[0065] In some embodiments, the control ABPCs are selected from one of the following: (a) 15G7; (b) 24D9; and (c) 29F1. In some embodiments, the control ABPCs are selected from one of the following: (a) hu15G7; (b) hu24D9; and (c) hu29F1.

[0066] In some embodiments, the ABPC includes a second ABD.

[0067] Also provided herein are kits comprising at least one dose of any of the pharmaceutical compositions described herein.

[0068] Further provided herein are antigen binding protein constructs (ABPCs) comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell, wherein: (a) the first ABD has a faster dissociation rate at a pH of about 4.0 to about 6.5 than at a pH of about 7.0 to about 8.0; or (b) the first ABD has a dissociation constant (K) of about 0.01 at a pH of about 4.0 to about 6.5. D ) than K at a pH of about 7.0 to about 8.0 D big.

[0069] In some embodiments, the first ABD comprises the HCV domain of one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises the LCV domain of one of the following: (a) the LCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the LCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the LCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0070] In some embodiments, the first ABD comprises one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate; and / or the LCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate.

[0071] In some embodiments, the HCV domain includes one of the following: (a) an HCV domain of 15G7, which includes SEQ ID NO:378 or a humanized version thereof (e.g., SEQ ID NO:382); (b) an HCV domain of 24D9, which includes SEQ ID NO:512 or a humanized version thereof (e.g., SEQ ID NO:516); and (c) an HCV domain of 29F1, which includes SEQ ID NO:572 or a humanized version thereof (e.g., SEQ ID NO:576). In some embodiments, the LCV domain comprises one of the following: (a) an LCV domain of 15G7 comprising SEQ ID NO:451 or a humanized version thereof (e.g., SEQ ID NO:455); (b) an LCV domain of 24D9 comprising SEQ ID NO:518 or a humanized version thereof (e.g., SEQID NO:522); and (c) an LCV domain of 29F1 comprising SEQ ID NO:577 or a humanized version thereof (e.g., SEQID NO:581).

[0072] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 573-575, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 573-575 are substituted with histidine, aspartic acid or glutamic acid. In some embodiments, the first LRRC15 binding domain comprises an LCV domain of one of the following: (a) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 519-521 are substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 578-580, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0073] In some embodiments, the first LRRC15 binding domain comprises one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: A total of one or more amino acid positions in NO:519-521 are substituted with histidine; and (c) an HCV domain, the HCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:573-575, respectively, optionally a total of one or more amino acid positions in SEQID NO:573-575 are substituted with histidine, aspartic acid or glutamic acid; and / or an LCV domain, the LCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:578-580, respectively, optionally a total of one or more amino acid positions in SEQID NO:578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0074] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises 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 HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises 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 HCV domain that is identical to the sequence shown in SEQ ID NO: The sequence shown in NO:572 or SEQ ID NO:576 is at least 90% identical, wherein the HCV domain includes aspartic acid (D) or glutamic acid (E) at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59. In some embodiments, the first ABD comprises an LCV domain, which is one of: (a) an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 LCV domain that is identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:579. The sequence shown in NO:581 is at least 90% identical, wherein the LCV domain comprises histidine, aspartic acid or glutamic acid at one or more positions in SEQID NO:577 or SEQ ID NO:581 selected from the group consisting of: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

[0075] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain comprises histidine at two or more positions in SEQ ID NO:378 or SEQ ID NO:382 comprising a pair selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516.

[0076] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises 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 shown in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises 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 HCV domain that is identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: NO:516, wherein the HCV domain comprises 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 an LCV domain, the LCV domain being at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 HCV domain, the HCV domain being at least 90% identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises histidine at one or more positions selected from the group consisting of: 35 and 97 in SEQ ID NO:572 or SEQ ID NO:576. NO:576 comprises aspartic acid (D) or glutamic acid (E) at one or more positions selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59; and / or a LCV domain, wherein the LCV domain is at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56 in SEQ ID NO:577 or SEQ ID NO:581.

[0077] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 512, SEQ ID NO: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: NO:653-695; and / or the first ABD includes an LCV domain of one of the following: (a) an LCV domain, the LCV domain having a sequence of one of the following: SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, and one of SEQ ID NOs:486-511; (b) an LCV domain, the LCV domain comprising a sequence of SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H) or SEQ ID NO:518 (with G97H); and (c) an LCV domain, the LCV domain comprising a sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and one of SEQ ID NOs:696-723.

[0078] In some embodiments, the first ABD includes one of the following: (a) an HCV domain comprising a sequence of one of SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain having a sequence of one of SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, wherein the first ABD does not include (i) an HCV domain of SEQ ID NO: 378 or SEQ ID NO: 382 and an LCV domain comprising a sequence that is not one of 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, and SEQ ID NO: NO:480-482; or (ii) an LCV domain comprising a sequence of SEQ ID NO:451 or SEQ ID NO:455 and an HCV domain comprising a sequence excluding one of the following: one of SEQ ID NOs:527-529, one of SEQ ID NOs:535-536, one of SEQ ID NOs:541-543, one of SEQ ID NOs:552, one of SEQ ID NOs:559-560, and SEQ ID NO:564; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO:512, SEQ ID NO:516, one of SEQ ID NOs:523-567, and one of SEQ ID NOs:568-571; and / or an LCV domain comprising a sequence of SEQ ID NO:518, SEQ ID NO:522, SEQ ID NO:524-528, SEQ ID NO:529-530; ID NO: 518 (with L35H substitution) or SEQ ID NO: 518 (with G97H substitution), wherein the first ABD does not include (i) a LCV domain comprising a sequence of SEQ ID NO: 512 or SEQ ID NO: 516 and a sequence that is not 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 excluding one of the following: one of SEQ ID NOs:527-529, one of SEQ ID NOs:535-536, one of SEQ ID NOs:541-543, SEQ ID NO:552, one of SEQ ID NOs:559-560, and SEQ ID NO:564; and (c) an HCV domain comprising the sequence of one of the following: SEQ ID NO:572, SEQ ID NO:576, one of SEQ ID NOs:582-624, and one of SEQ ID NOs:653-695; and / or an LCV domain comprising the sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and SEQ ID NO:696-723, wherein the first ABD does not include (i) an HCV domain comprising a sequence of SEQ ID NO:572 or SEQ ID NO:576 and an LCV domain comprising a sequence that is not one of the following: SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:632, SEQ ID NO:638, and SEQ ID NO:642; or (ii) an LCV domain comprising a sequence of SEQ ID NO:577 or SEQ ID NO:581 and an HCV domain comprising a sequence that is not one of the following: SEQ ID NO:587, SEQ ID NO:598, SEQ ID NO:611, and SEQ ID NO:672. ;

[0079] In some embodiments, after the ABPC is internalized by the target mammalian cell, the ABPC is degraded in the target mammalian cell. In some embodiments, the ABPC comprises a conjugated toxin, radioisotope, drug or small molecule.

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

[0081] In some embodiments, the composition comprising the ABPC provides an increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 20% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 50% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 2-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 5-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0082] In some embodiments, the composition comprising the ABPC provides an increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the ABPC provides at least a 20% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 50% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 2-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 5-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0083] In some embodiments, the composition comprising the ABPCs causes less of a decrease in the level of LRRC15 presented on the surface of the target mammalian cell than a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPCs does not cause a detectable decrease in the level of LRRC15 presented on the surface of the target mammalian cell.

[0084] Also provided herein are antigen binding protein constructs (ABPCs) comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug, or small molecule, wherein: (a) the dissociation rate of the first ABD at 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 greater than the dissociation rate at a pH of about 7.0 to about 8.0. D ) is greater than K at a pH of about 7.0 to about 8.0 D ; and (b) the composition provides one or more of the following: increased toxin release in the target mammalian cells compared to a composition comprising the same amount of a control ABPC; increased target mammalian cell killing compared to a composition comprising the same amount of a control ABPC; and increased endolysosomal delivery in the target mammalian cells compared to a composition comprising the same amount of a control ABPC.

[0085] In some embodiments, the first ABD comprises the HCV domain of one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid. In some embodiments, the first ABD comprises the LCV domain of one of the following: (a) the LCV domain of 15G7, which optionally has one or more amino acids substituted with histidine; (b) the LCV domain of 24D9, which optionally has one or more amino acids substituted with histidine; and (c) the LCV domain of 29F1, which optionally has one or more amino acids substituted with histidine, aspartic acid, or glutamic acid.

[0086] In some embodiments, the first ABD comprises one of the following: (a) the HCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 15G7, which optionally has one or more amino acids substituted by histidine; (b) the HCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and / or the LCV domain of 24D9, which optionally has one or more amino acids substituted by histidine; and (c) the HCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate; and / or the LCV domain of 29F1, which optionally has one or more amino acids substituted by histidine, aspartic acid or glutamate.

[0087] In some embodiments, the HCV domain includes one of the following: (a) an HCV domain of 15G7, which includes SEQ ID NO:378 or a humanized version thereof (e.g., SEQ ID NO:382); (b) an HCV domain of 24D9, which includes SEQ ID NO:512 or a humanized version thereof (e.g., SEQ ID NO:516); and (c) an HCV domain of 29F1, which includes SEQ ID NO:572 or a humanized version thereof (e.g., SEQ ID NO:576). In some embodiments, the LCV domain comprises one of the following: (a) an LCV domain of 15G7 comprising SEQ ID NO:451 or a humanized version thereof (e.g., SEQ ID NO:455); (b) an LCV domain of 24D9 comprising SEQ ID NO:518 or a humanized version thereof (e.g., SEQID NO:522); and (c) an LCV domain of 29F1 comprising SEQ ID NO:577 or a humanized version thereof (e.g., SEQID NO:581).

[0088] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and (c) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 573-575, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 573-575 are substituted with histidine, aspartic acid or glutamic acid. In some embodiments, the first LRRC15 binding domain comprises an LCV domain of one of the following: (a) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 519-521 are substituted with histidine; and (c) an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 578-580, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0089] In some embodiments, the first LRRC15 binding domain comprises one of the following: (a) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379-381, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 379-381 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452-454, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 452-454 are substituted with histidine; (b) an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 513-515, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: 513-515 are substituted with histidine; and / or an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 519-521, respectively, optionally wherein a total of one or more amino acid positions in SEQ ID NOs: A total of one or more amino acid positions in NO:519-521 are substituted with histidine; and (c) an HCV domain, the HCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:573-575, respectively, optionally a total of one or more amino acid positions in SEQID NO:573-575 are substituted with histidine, aspartic acid or glutamic acid; and / or an LCV domain, the LCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NO:578-580, respectively, optionally a total of one or more amino acid positions in SEQID NO:578-580 are substituted with histidine, aspartic acid or glutamic acid.

[0090] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises 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 HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: 516, wherein the HCV domain comprises 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 HCV domain that is identical to the sequence shown in SEQ ID NO: The sequence shown in NO:572 or SEQ ID NO:576 is at least 90% identical, wherein the HCV domain includes aspartic acid (D) or glutamic acid (E) at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59. In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:582. NO:577 or SEQ ID NO:581 comprises histidine, aspartic acid or glutamic acid at one or more positions selected from the group consisting of: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

[0091] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain comprises histidine at two or more positions in SEQ ID NO:378 or SEQ ID NO:382 comprising a pair selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain comprises histidine at one or more positions in SEQ ID NO:512 or SEQ ID NO:516.

[0092] In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain, which is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain comprises histidine at two or more positions in SEQ ID NO:451 or SEQ ID NO:455 comprising a 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, which is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises histidine at one or more positions in SEQ ID NO:518 or SEQ ID NO:522.

[0093] In some embodiments, the first ABD comprises one of: (a) an HCV domain that is at least 90% identical to the sequence shown in SEQ ID NO: 378 or 382, ​​wherein the HCV domain comprises 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 shown in SEQ ID NO: 451 or SEQ ID NO: 455, wherein the LCV domain comprises 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 HCV domain that is identical to the sequence shown in SEQ ID NO: 512 or SEQ ID NO: NO:516, wherein the HCV domain comprises 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 an LCV domain, the LCV domain being at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain comprises 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 HCV domain, the HCV domain being at least 90% identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain comprises histidine at one or more positions selected from the group consisting of: 35 and 97 in SEQ ID NO:572 or SEQ ID NO:576. NO:576 comprises aspartic acid (D) or glutamic acid (E) at one or more positions selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59; and / or a LCV domain, wherein the LCV domain is at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56 in SEQ ID NO:577 or SEQ ID NO:581.

[0094] In some embodiments, the first ABD comprises an HCV domain of one of the following: (a) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 378, one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 512, SEQ ID NO: 516, one of SEQ ID NOs: 523-567, and one of SEQ ID NOs: 568-571; and (c) an HCV domain comprising a sequence of one of the following: SEQ ID NO: 572, SEQ ID NO: 576, one of SEQ ID NOs: 582-624, and one of SEQ ID NOs: 653-695. In some embodiments, the first ABD comprises an LCV domain of one of the following: (a) an LCV domain having a sequence of one of the following: SEQ ID NO:451, SEQ ID NO:455, one of SEQ ID NOs:456-485, and one of SEQ ID NOs:486-511; (b) an LCV domain comprising a sequence of one of the following: SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H), or SEQ ID NO:518 (with G97H); and (c) an LCV domain comprising a sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and one of SEQ ID NOs:696-723.

[0095] In some embodiments, the first ABD includes one of the following: (a) an HCV domain comprising a sequence of one of SEQ ID NO: 378, SEQ ID NO: 382, ​​one of SEQ ID NOs: 383-424, and one of SEQ ID NOs: 425-450, and / or an LCV domain having a sequence of one of SEQ ID NO: 451, SEQ ID NO: 455, one of SEQ ID NOs: 456-485, and one of SEQ ID NOs: 486-511, wherein the first ABD does not include (i) an HCV domain of SEQ ID NO: 378 or 382 and an LCV domain comprising a sequence that is not one of 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, and SEQ ID NO: NO:480-482; or (ii) an LCV domain comprising the sequence of SEQ ID NO:451 and an HCV domain comprising a sequence that does not include one of the following: one of SEQ ID NO:527-529, one of SEQ ID NO:535-536, one of SEQ ID NO:541-543, one of SEQ ID NO:552, one of SEQ ID NO:559-560, and SEQ ID NO:564; (b) an HCV domain comprising a sequence of one of the following: SEQ ID NO:512, SEQ ID NO:516, one of SEQ ID NO:523-567, and one of SEQ ID NO:568-571; and / or an LCV domain comprising a sequence of SEQ ID NO:518, SEQ ID NO:522, SEQ ID:518 (with L35H), or SEQ ID NO:5 ID NO: 518 (with G97H), wherein the first ABD does not include (i) a sequence comprising SEQ ID NO: 512 or SEQ ID NO: 516 and an LCV domain comprising a sequence that is not 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 excluding one of the following: one of SEQ ID NOs:527-529, one of SEQ ID NOs:535-536, one of SEQ ID NOs:541-543, SEQ ID NO:552, one of SEQ ID NOs:559-560, and SEQ ID NO:564; and (c) an HCV domain comprising the sequence of one of the following: SEQ ID NOs:572, 576, or 582-624 or 653-695; and / or an LCV domain comprising the sequence of one of the following: SEQ ID NO:577, SEQ ID NO:581, one of SEQ ID NOs:625-652, and SEQ ID NO:696-723, wherein the first ABD does not include (i) an HCV domain comprising a sequence of SEQ ID NO:572 or SEQ ID NO:576 and an LCV domain comprising a sequence that is not one of the following: SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:632, SEQ ID NO:638, and SEQ ID NO:642; or (ii) an LCV domain comprising a sequence of SEQ ID NO:577 or SEQ ID NO:581 and an HCV domain comprising a sequence that is not one of the following: SEQ ID NO:587, SEQ ID NO:598, SEQ ID NO:611, and SEQ ID NO:672. ;

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

[0097] In some embodiments, the composition comprising the ABPC provides an increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 20% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 50% increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 2-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 5-fold increase in killing of the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0098] In some embodiments, the composition comprising the ABPC provides an increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 20% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 50% increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 2-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPC provides at least a 5-fold increase in endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

[0099] In some embodiments, the composition comprising the ABPCs causes less reduction in the level of LRRC15 presented on the surface of the target mammalian cell than a composition comprising the same amount of a control ABPC. In some embodiments, the composition comprising the ABPCs does not cause a detectable reduction in the level of LRRC15 presented on the surface of the target mammalian cell. In some embodiments, the target mammalian cell is a cancer cell.

[0100] In some embodiments, the ABD has an off-rate at a pH of about 4.0 to about 6.5 that is at least 10% faster than the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the off-rate of the ABD at a pH of about 4.0 to about 6.5 is at least 3 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the off-rate of the ABD at a pH of about 4.0 to about 6.5 is at least 10 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. In some embodiments, the ABD has an off-rate at a pH of about 4.0 to about 6.5 that is at least 10 times the off-rate of the ABD at a pH of about 7.0 to about 8.0. D than the K of the ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the ABD has a K of about 10% greater than the ABD at a pH of about 4.0 to about 6.5. D is the K of the ABD at a pH of about 7.0 to about 8.0 D In some embodiments, the ABD has a K of at least 3 times greater than that of the ABD at a pH of about 4.0 to about 6.5. D is the K of the ABD at a pH of about 7.0 to about 8.0 D At least 10 times.

[0101] In some embodiments, the ABPC is cytotoxic or cytostatic to the target mammalian cells.

[0102] In some embodiments, the ABPCs are cross-reactive with non-human primate LRRC15 and human LRRC15. In some embodiments, the ABPCs are cross-reactive with non-human primate LRRC15, human LRRC15, and one or both of rat LRRC15 and mouse LRRC15. In some embodiments, the ABPCs are 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 a cell from an old world monkey.

[0103] 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 a scFv. In some embodiments, the ABPC is a BiTe, (scFv) 2 , nanobody, nanobody-HSA, DART, TandAb, sc bifunctional antibody, sc bifunctional antibody-CH3, scFv-CH-CL-scFv, HSAbody, sc bifunctional antibody-HSA or tandem scFv.

[0104] In some embodiments, the ABPC comprises two or more polypeptides. In some embodiments, the ABPC is selected from the group consisting of antibodies, VHH-scAb, VHH-Fab, double scFab, F(ab') 2 , bifunctional antibody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common light chain of knob and hole, knob and 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, bifunctional antibody-CH3, trifunctional antibody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab') 2 -scFv 2 , scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, intrabody, docking lock antibody, ImmTAC, IgG-IgG conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAB and PROTAC.

[0105] In some embodiments, the ABPC or the ABD comprises a proteolysis targeting antibody (PROTAB) (as described, for example, in Marei et al., "Antibody targeting of E3 ubiquitin ligases for receptor degradation". Nature, October 6, 2022). In some embodiments, PROTABs or PROTACs can link cell surface E3 ubiquitin ligases to transmembrane proteins, causing target degradation both in vitro and in vivo.

[0106] In some embodiments, at least one polypeptide of the ABPC is conjugated to the toxin, the radioisotope, the drug, or the small molecule via a cleavable linker. In some embodiments, at least one polypeptide of the ABPC is conjugated to the toxin, the radioisotope, the drug, or the small molecule via a non-cleavable linker.

[0107] In some embodiments, the in vivo half-life of the ABPC is increased compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 5% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 10% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 30% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 50% to about 95% compared to the in vivo half-life of the control ABPC. In some embodiments, the in vivo half-life of the ABPC is increased by about 70% to about 95% compared to the in vivo half-life of the control ABPC.

[0108] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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 3 times 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 D ) is greater than K at a pH of about 7.0 to about 8.0 D No more than 3 times larger.

[0109] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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 2 times 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 D ) is greater than K at a pH of about 7.0 to about 8.0 D No more than 2 times larger.

[0110] In some embodiments, the control ABPC is capable of specifically binding to LRRC15 or an LRRC15 epitope presented 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) of the first ABD of the control ABPC at a pH of about 4.0 to about 6.5 is D ) is greater than K at a pH of about 7.0 to about 8.0 D No more than 1 times larger.

[0111] In some embodiments, the control ABPC is selected from one of the following: (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.

[0112] In some embodiments, the ABPC includes a second ABD.

[0113] Also provided herein are kits comprising at least one dose of any of the ABPCs described herein.

[0114] Also provided herein is a method for treating cancer characterized by a population of cancer cells, cancer associated fibroblasts (CAFs) and / or stromal cells having LRRC15 or an epitope of LRRC15 presented 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 said cancer cells, CAFs and / or stromal cells.

[0115] Also provided herein is a method for reducing the volume of a tumor in a subject, wherein the tumor is characterized by a population of cancer cells, CAFs and / or stromal cells having LRRC15 or an epitope of LRRC15 presented on its 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 said cancer cells, CAFs and / or stromal cells.

[0116] Also provided herein is a method of inducing cell death in cancer cells, CAFs and / or stromal cells in a subject, wherein the cancer cells, the CAFs and / or the stromal cells have LRRC15 or an epitope of LRRC15 presented on their surface, wherein the method comprises: administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having a cancer characterized by having a population of the cancer cells, the CAFs and / or the stromal cells.

[0117] In some embodiments, the cancer is a primary tumor or metastasis. In some embodiments, the cancer is a non-T cell infiltrating tumor. In some embodiments, the cancer is a T cell infiltrating tumor.

[0118] Also provided herein is a method of reducing the risk of metastasis in a subject with cancer or reducing the risk of additional metastasis, wherein the cancer is characterized by a population of cancer cells, CAFs, and / or stromal cells having LRRC15 or an epitope of LRRC15 presented on its 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 the cancer cells, CAFs, and / or stromal cells. In some embodiments, the cancer is a sarcoma, e.g., leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0119] As used herein, the term "antigen binding protein construct" is: (i) a single polypeptide comprising at least one ABD; or (ii) a complex of two or more polypeptides (e.g., the same polypeptide or different polypeptides) that together form at least one ABD. Non-limiting examples and aspects of antigen binding protein constructs are described herein. Additional examples and aspects of antigen binding protein constructs are known in the art.

[0120] "Multi-specific antigen-binding protein constructs" are antigen-binding protein constructs that include two or more different ABDs that specifically bind to two or more different epitopes together. The two or more different epitopes can be epitopes on the same antigen (e.g., a single polypeptide present on a cell surface) or different antigens (e.g., different proteins present on the same cell surface or on different cell surfaces). In some aspects, the antigen is present on the cell surface. In some aspects, the multi-specific antigen-binding protein construct binds two different epitopes (i.e., a "bispecific antigen-binding protein construct"). In some aspects, the multi-specific antigen-binding protein construct binds three different epitopes (i.e., a "trispecific antigen-binding protein construct"). In some aspects, the multi-specific antigen-binding protein construct binds four different epitopes (i.e., a "tetraspecific antigen-binding protein construct"). In some aspects, the multi-specific antigen-binding protein construct binds five different epitopes (i.e., a "five-specific antigen-binding protein construct"). Each binding specificity can exist at any suitable valence. Non-limiting examples of multi-specific antigen-binding protein constructs are described herein.

[0121] "ABD" is one or more protein domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some instances, the ABD may bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the ABD may be an antibody or a fragment thereof. In some embodiments, the ABD may include an alternative scaffold. Non-limiting examples of ABDs are described herein. Additional examples of ABDs are known in the art. In some instances, the ABD may bind to a single antigen.

[0122] The term "antibody" is used in the broadest sense herein and includes certain types of immunoglobulin molecules that include one or more ABDs that specifically bind to an antigen or epitope. Antibodies particularly include, for example, complete antibodies (e.g., complete 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.

[0123] 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, where molecules internalized by cell-mediated internalization processes (e.g., pinocytosis, micropinocytosis, receptor-mediated endocytosis, and / or phagocytosis) are sorted.

[0124] Once the endosomes in the endosomal / lysosomal pathway are purified or isolated, the target protein (e.g., an antigen binding protein construct described herein) can be assayed using methods known in the art (ELISA, Western blot, immunofluorescence, and immunoprecipitation followed by determination of protein concentration) and can be used to determine the concentration or relative level of the target protein in the endosome. Alternatively, immunofluorescence microscopy can be used using a detectably labeled antibody (e.g., a fluorophore-labeled antibody, a dye-labeled antibody, or a GFP-labeled antibody, e.g., CellLight TM Endosomes in the endosomal / lysosomal pathway can be imaged by combining early endosome-GFP) and a fluorophore-labeled antibody that specifically binds to a target protein (e.g., an antigen binding protein construct), and the level of the target protein in the endosome can be determined by quantifying the overlap in the fluorescence emission of the two different antibodies.

[0125] The phrase "endolysosomal delivery" refers to the rate of accumulation of an antigen binding protein construct (e.g., any antigen binding protein construct described herein) in the endosomal / lysosomal pathway of a mammalian cell (e.g., any exemplary target mammalian cell described herein) over time or the total rate of accumulation at a particular time point.

[0126] An exemplary method for calculating the increase in endolysosomal delivery of a pH-engineered ABPC variant compared to its corresponding starting ABPC from the cell fluorescence data is to measure the mean fluorescence intensity of the variant minus the mean fluorescence intensity of a non-binding IgG control and then divide the whole by the ratio of the mean fluorescence intensity of the variant's corresponding starting ABPC minus the mean fluorescence intensity of the IgG control.

[0127] Exemplary assays for measuring endolysosomal delivery of any ABPC described herein include those involving labeling the ABPC with a fluorescent dye, then incubating the labeled ABPC with cells and measuring cell fluorescence as an indicator of ABPC endolysosomal delivery (e.g., as generally described in Wustner, Traffic 7(6): 699-715, 2006). Alternatively, a pH-sensitive dye that preferentially fluoresces at an acidic pH rather than a neutral pH can be used to label any ABPC described herein, which can then be incubated with cells and measuring cell fluorescence as an indicator of ABPC delivery to an acidic endolysosomal compartment.

[0128] The term "population" when used before a noun means two or more of the specific noun. For example, the phrase "a population of cancer cells" means "two or more cancer cells." Non-limiting examples of cancer cells are described herein.

[0129] The phrase "cytostatic to a cell" refers to a direct or indirect reduction in the 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 a cell (e.g., a cancer cell). In some instances, an agent that is cytostatic to a cell can reduce the number of cells in the S phase in a cell population (compared to the number of cells in the S phase in the cell population prior to contact with the agent). In some instances, an agent that is cytostatic to a cell can reduce the percentage of cells in the 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 the S phase in the cell population prior to contact with the agent).

[0130] The phrase "cytotoxic to cells" refers to inducing, directly or indirectly, the death (eg, necrosis or apoptosis) of cells (eg, mammalian cells, such as cancer cells).

[0131] "Affinity" refers to the strength of the sum of non-covalent interactions between an antigen binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise specified, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between a member of an ABD and an antigen or epitope. The affinity of a molecule X for its partner Y can be expressed in terms of the dissociation equilibrium constant (K D ). Affinity can be measured by common methods known in the art (including those described herein). Affinity can be measured, for example, using surface plasmon resonance (SPR) technology (e.g., ) or biolayer interferometry (e.g. Additional methods for determining affinity for an ABD and its corresponding antigen or epitope are known in the art.

[0132] The term "epitope" means a portion of an antigen that is specifically bound by an ABD through a set of physical interactions between: (i) all monomers on the portion of the ABD that specifically binds to the antigen (e.g., individual amino acid residues, sugar side chains, and post-translationally modified amino acid residues), and (ii) all monomers on the portion of the antigen that is specifically bound by the ABD (e.g., individual amino acid residues, sugar side chains, and post-translationally modified amino acid residues). An epitope may, for example, consist of surface accessible amino acid residues, sugar side chains, phosphorylated amino acid residues, methylated amino acid residues, and / or acetylated amino acid residues and may have specific three-dimensional structural features and specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that binding to the former, rather than binding to the latter, may be lost in the presence of a denaturing solvent. In some embodiments, an epitope is defined by a linear amino acid sequence of at least about 3 to 6 amino acids or about 10 to 15 amino acids. In some embodiments, an epitope refers to a portion of a full-length protein defined by a three-dimensional structure (e.g., protein folding) or a portion thereof. In some embodiments, an epitope is defined by a discontinuous amino acid sequence that is aggregated together via protein folding. In some embodiments, an epitope is defined by a discontinuous amino acid sequence 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 defining the epitope may not be critical to the three-dimensional structure of the epitope. Conformational epitopes can be determined and screened using assays that compare the binding of an antigen binding protein construct to a denatured form of the antigen in order to generate a linear epitope. An epitope can include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding.

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

[0134] The term "paratope" means a portion of an ABD that specifically binds to an antigen through a set of physical interactions between: (i) all monomers on the portion of the ABD that specifically binds to the antigen (e.g., separate amino acid residues, sugar side chains, post-translationally modified amino acid residues), and (ii) all monomers on the portion of the antigen that is specifically bound by the ABD (e.g., separate amino acid residues, sugar side chains, post-translationally modified amino acid residues). A paratope can, for example, consist of surface accessible amino acid residues and can have specific three-dimensional structural properties and specific charge properties. In some embodiments, a paratope refers to a portion of a full-length ABD defined by a three-dimensional structure (e.g., protein folding) or a portion thereof. In some embodiments, a paratope is defined by a discontinuous amino acid sequence that is aggregated together via protein folding. In some embodiments, an epitope is defined by a discontinuous amino acid sequence that is aggregated 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 a paratope may not be critical to the three-dimensional structure of a paratope. A paratope can include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding.

[0135] Methods for identifying the paratope to which the ABD specifically binds are known in the art, such as structure-based analysis (e.g., X-ray crystallography, NMR and / or electron microscopy) (e.g., based on the ABD and / or the ABD-antigen complex) and / or mutagenesis-based analysis (e.g., alanine scanning mutagenesis, glycine scanning mutagenesis, and homology scanning mutagenesis), wherein mutants are measured in binding assays with the binding partner, many of which are known in the art.

[0136] The phrase "present on the surface of a mammalian cell" means an antigen that is (1) physically attached to 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-myristylated protein, or an S-palmitoylated protein) or (2) stably associated with its cognate receptor, wherein the cognate receptor is physically attached to the plasma membrane of a mammalian cell (e.g., a ligand that binds to its cognate receptor, wherein 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 blotting.

[0137] The phrase "control ABPC" or "control antigen binding protein construct" means (i) an ABPC capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a mammalian cell (e.g., a target mammalian cell), wherein one or both of the following are true: (a) the first ABD has a higher off rate at a pH of about 4.0 to about 6.5 (e.g., any of the subranges of this range described herein) than at a pH of about 7.0 to about 8.0 (e.g., any of the subranges of this range described herein); or the 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 the 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 ) 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 and / or (iii) 29F1; (i) 15G7; (ii) 24D9; and / or (iii) 29F1.

[0138] The term "extracellular space" means the fluid outside the plasma membrane of a mammalian cell. When the mammalian cell is in vitro, the extracellular space can be a liquid culture medium. When the mammalian cell is in vivo, the extracellular space can be, for example, plasma, serum, blood, interstitial fluid, or lymph.

[0139] The term "endolysosomal space" refers to the fluid enclosed by the vesicles and organelles that make up the endosomal / lysosomal pathway in mammalian cells.

[0140] The phrase "reduced levels" or "decreased levels" 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%) compared to a reference level or value.

[0141] The term "cell killing efficacy" refers to the ability of an agent (e.g., any ABPC described herein) to directly or indirectly induce apoptosis and / or necrosis of mammalian cells (e.g., cancer cells) as measured over time or at a rate at a relevant time point. Methods for determining the cell killing efficacy of cells are known in the art (e.g., trypan blue staining, microscopy, fluorescence-assisted cell sorting, and assays for detecting apoptosis markers (e.g., annexin V)). In a non-limiting example, the cell killing efficacy can be measured in the following manner, such as by cell killing at a single concentration of the agent, by the IC50 of the agent (i.e., the concentration of the agent that achieves half the maximum cell killing efficacy), or by the ratio of the dissociation constant KD of the agent on mammalian cells divided by its IC50. In some non-limiting examples, the IC50 and / or KD ratios described herein are compared to the ratios of the control ABPCs (as defined herein), and it is optionally demonstrated that the ABPCs described herein have a higher cell killing efficacy than the control ABPCs.

[0142] The term "toxin release" refers to the ability of a mammalian cell (e.g., a non-cancerous mammalian cell or a cancer cell) to internalize (e.g., via pinocytosis and / or receptor-mediated endocytosis) any ABPC described herein (e.g., any ABPC described herein or a control ABPC) conjugated to a toxin, as measured over time or at a rate at a specific time point, and subsequently release the toxin conjugated to the ABPC. 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 toxins.

[0143] The phrase "target cell" or "target mammalian cell" or "mammalian target cell" means a mammalian cell having at least one LRRC15 present on its surface. In some examples, the mammalian target cell can be a cancer cell. In some embodiments of the target mammalian cell, there can be about the following totals (each ± ​​about 10%) of LRRC15 present on the cytoplasmic membrane of the target mammalian cell: 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, 1-40,000, 1-35,000, 1-30,000, 1-25,000, 1-20,000, 1-15,000, 1-10,000, 1-7,500, 1-5,000, 1-4,000, 1-3,000, 1-2,000, 1-1,000, 1-500, 1-100, 1-50 or 1-10, or any numerical range in the numerical ranges recited in U.S. Patent Application No. 2022 / 0281984, which is incorporated herein in its entirety.

[0144] The phrase "antigen density" means the amount of LRRC15 present on the surface of a target mammalian cell or the average amount of LRRC15 on the surface of a population of target mammalian cells of a particular type. "Antigen density" can be measured using, for example, QuantiBRITE TM Bead kits or radiolabeled (e.g., BD Biosciences PE Phycoerythrin Fluorescence Quantitation Kit, catalog number 340495).

[0145] The phrase "amino acid is substituted with histidine" means that an amino acid residue in a reference polypeptide sequence that is not histidine is substituted with histidine. Non-limiting methods for substituting amino acid residues in a reference polypeptide with histidine are described herein. Additional methods for substituting amino acid residues in a reference polypeptide with histidine are known in the art.

[0146] The phrase "amino acid is substituted with alanine" means that an amino acid residue in a reference polypeptide sequence that is histidine is substituted 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.

[0147] 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 the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may 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 the event of a conflict, the present specification (including definitions) shall prevail.

[0148] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] FIG1A shows the binding of 15G7 and its HC variants to LRRC15 by biolayer interferometry (BLI). For each individual figure, the antigen binding protein was captured on an anti-human Fc biosensor and associated with LRRC15 at pH 7.4. Dissociation was performed at pH 7.4 (black) or pH 5.4 (grey). Primary X-axis scale = 100 seconds, with left dashed vertical line = 0 seconds. Primary Y-axis scale = 0.5 nm, and the octet plot shows MYT5712 and MYT6080-MYT6121.

[0150] Figure 1B Binding of 15G7 and its LC variants to LRRC15 is shown by BLI. Primary Y-axis scale = 0.2 nm and octet plots show MYT6122-MYT6151.

[0151] Figure 1C Binding of humanized 15G7 and its HC variants to LRRC15 is shown by BLI. Primary Y-axis scale = 0.5 nm and octet plots show MYT8387-MYT8411.

[0152] Figure 1D Binding of humanized 15G7 and its LC variants to LRRC15 is shown by BLI. Primary Y-axis scale = 0.2 nm and octet plots show MYT8413-MYT8437.

[0153] FIG. 1E shows the combined data of FIG. 1A plotted to maximize vertical spacing.

[0154] Figure 1F shows the plotted lines to maximize vertical spacing. Figure 1B of combined data.

[0155] Figure 1G shows the plotted lines to maximize vertical spacing. Figure 1C Combined data.

[0156] Figure 1H Shows the Figure 1D Combined data plotted to maximize the vertical spacing.

[0157] Figure 2A shows the binding of 24D9 and its HC variants to LRRC15 by BLI. Primary Y-axis scale = 0.2 nm, and the octet plot shows MYT5742 and MYT7881 - MYT7925.

[0158] Figure 2B shows the combined data of Figure 2A plotted to maximize the vertical spacing.

[0159] Figure 3A The binding of 24D9 and its LC variants to LRRC15 is shown by BLI. Primary Y-axis scale = 0.2 nm, and the octet plot shows MYT7926 - MYT7958.

[0160] Figure 3B Shows the Figure 3A Combined data plotted to maximize the vertical spacing.

[0161] Figure 4A shows the binding of humanized 29F1 and its HC aspartic acid variants to LRRC15 by BLI. Primary Y-axis scale = 0.1 nm, and the octet plot shows MYT8094 and MYT9464 - MYT9506.

[0162] Figure 4B shows the combined data of Figure 4A plotted to maximize the vertical spacing.

[0163] Figure 5A The binding of humanized 29F1 and its LC aspartic acid variants to LRRC15 is shown by BLI. Primary Y-axis scale = 0.1 nm, and the octet plot shows MYT9507 - MYT9534.

[0164] Figure 5B Shows the Figure 5A Combined data plotted to maximize the vertical spacing.

[0165] Figure 6A shows the binding of humanized 29F1 and its HC glutamic acid variants to LRRC15 by BLI. Primary Y-axis scale = 0.2 nm, and the octet plot shows MYT9684 - MYT9726.

[0166] Figure 6B shows the combined data of Figure 6A plotted to maximize the vertical spacing.

[0167] Fig. 7ABinding of humanized 29F1 and its LC glutamate variant to LRRC15 is shown by BLI. Primary Y-axis scale = 0.2 nm and octet plots show MYT9727-MYT9754.

[0168] Figure 7B shows the plotted lines to maximize vertical spacing. Fig. 7A of combined data.

[0169] Figure 8 Shown are graphs of median tumor volumes (TV) at indicated days following administration of indicated test articles or controls to SA4033 tumor-bearing mice.

[0170] Fig. 9 Graphs are presented of the median TV at the indicated days following administration of the indicated test article or control in the CTG-0241 model.

[0171] Fig. 10A Shown are graphs of median TV at indicated days following administration of test article or control in the SA3851 model.

[0172] Fig. 10B Graphs are presented of the median TV at the indicated days following administration of the indicated test article or control in the SA4040 model.

[0173] Fig. 10C Shown are graphs of median TV at indicated days following administration of indicated test article or control in the SA4109 model. DETAILED DESCRIPTION

[0174] Provided herein are antigen binding protein constructs (ABPCs) comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 presented 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 ) than K at a pH of about 7.0 to about 8.0 D In some examples of these ABPCs, after the ABPC is internalized by the target mammalian cell, the ABPC is degraded in the target mammalian cell. Some examples of any ABPC described herein may further include a conjugated toxin, radioisotope, drug, or small molecule (e.g., fluorophore or dye).

[0175] Also provided is an antigen binding protein construct (ABPC), comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell; and a conjugated toxin, a radioisotope, a drug or a small molecule, 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 the dissociation constant (K) of the first ABD at a pH of about 4.0 to about 6.5 is D ) than K at a pH of about 7.0 to about 8.0 D and (b) the composition comprising the ABPC provides one or more (e.g., two or three) of the following: increased toxin release in the target mammalian cells (e.g., a detectable increase) compared to a composition comprising the same amount of a control ABPC; increased target mammalian cell killing (e.g., a detectable increase) compared to a composition comprising the same amount of a control ABPC; and increased endolysosomal delivery (e.g., a detectable increase) in the target mammalian cells (e.g., a detectable increase) compared to a composition comprising the same amount of a control ABPC.

[0176] In some instances, the first ABD includes the HCV domain of 15G7, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine. In some instances, the first ABD includes the LCV domain of 15G7, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine. In some instances, the first ABD includes the HCV domain of 15G7, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine; and the LCV domain of 15G7, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine. In some instances, 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.

[0177] In some examples, the first ABD comprises an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379, 380 and 381, respectively, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions in total substituted with histidine in common in SEQ ID NOs: 379-381. In some examples, the first ABD comprises an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452, 453 and 454, respectively, optionally with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions in total substituted with histidine in common in SEQ ID NOs: 452-454. In some instances, the first ABD comprises: an HCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 379, 380 and 381, respectively, optionally in which a total of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions in NOs: 379-381 are commonly substituted with histidine; and an LCV domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 452, 453 and 454, respectively, optionally in which 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 are commonly substituted with histidine.

[0178] In some instances, the first ABD includes an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​optionally wherein the HCV domain includes 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 includes an LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, optionally wherein the LCV domain includes 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:451 or SEQ ID NO:455 selected from the group consisting of: 29, 30, 32, 34, 50, 92, 93, 95, 96, and 97. In some examples, the first ABD includes an HCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO: 378 or 382, ​​optionally wherein the HCV domain includes 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; and a LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO: 451 or SEQ ID NO: 455, optionally wherein the LCV domain is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO: 451 or SEQ ID NO: NO:455 includes 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.

[0179] In some instances, 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 shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes 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.

[0180] Table 1. Exemplary combinations of amino acid positions in SEQ ID NO: 378 or SEQ ID NO: 382 that can be substituted with histidine

[0181]

[0182] In some instances, the LCV domain includes an LCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes 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.

[0183] Table 2. Exemplary combinations of amino acid positions in SEQ ID NO: 451 or SEQ ID NO: 455 that can be substituted with histidine

[0184]

[0185]

[0186] In some instances, the first ABD includes an HCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes histidine at any combination of 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 shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:451 or SEQ ID NO:455 listed in Table 2.

[0187] In some instances, the first ABD comprises an LCV domain comprising the sequence shown in SEQ ID NO:451 or SEQ ID NO:455; and an HCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes 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.

[0188] In some instances, the first ABD comprises an LCV domain that is ≥90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 29 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0189] In some instances, the first ABD comprises an LCV domain that is ≥90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 30 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is ≥90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0190] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes histidine at position 32 in SEQ ID NO:451 or SEQ ID NO:455; and an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0191] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 34 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0192] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 50 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0193] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 92 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0194] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 93 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0195] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 95 in SEQ ID NO:451 or SEQ ID NO:455; and an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0196] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 96 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0197] In some instances, the first ABD comprises an LCV domain that is at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCV domain includes a histidine at position 97 in SEQ ID NO:451 or SEQ ID NO:455; and a HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:378 or 382, ​​wherein the HCV domain includes a histidine at any combination of specific combinations of one or more amino acid positions in SEQ ID NO:378 or 382 listed in Table 1.

[0198] In some examples, the first ABD comprises 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.

[0199] In some examples, the first ABD includes 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 includes 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.

[0201] In some examples, the first ABD includes 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.

[0202] In some examples, the first ABD includes 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.

[0203] In some examples, the first ABD includes 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.

[0204] In some examples, the first ABD includes 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.

[0205] In some examples, the first ABD includes 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.

[0206] In some examples, the first ABD includes 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.

[0207] In some examples, the first ABD includes 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.

[0208] In some examples, the first ABD includes 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.

[0209] In some examples, the first ABD includes 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.

[0210] In some examples, the first ABD includes 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.

[0211] In some examples, the first ABD includes 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.

[0212] In some examples, the first ABD includes 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.

[0213] In some examples, the first ABD includes 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.

[0214] In some examples, the first ABD includes 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.

[0215] In some examples, the first ABD includes 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.

[0216] In some examples, the first ABD includes 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.

[0217] In some examples, the first ABD includes 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.

[0218] In some examples, the first ABD includes 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.

[0219] In some examples, the first ABD includes 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.

[0220] In some examples, the first ABD includes 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.

[0221] In some examples, the first ABD includes 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.

[0222] In some examples, the first ABD includes 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.

[0223] In some examples, the first ABD includes 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.

[0224] In some examples, the first ABD includes 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.

[0225] In some examples, the first ABD includes 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.

[0226] In some examples, the first ABD includes 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.

[0227] In some examples, the first ABD includes 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.

[0228] In some examples, the first ABD includes 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.

[0229] In some examples, the first ABD includes 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.

[0230] In some examples, the first ABD includes 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.

[0231] In some examples, the first ABD includes 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.

[0232] In some examples, the first ABD includes 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.

[0233] In some examples, the first ABD includes 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.

[0234] In some examples, the first ABD includes 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.

[0235] In some examples, the first ABD includes 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.

[0236] In some examples, the first ABD includes 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.

[0237] In some examples, the first ABD includes 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.

[0238] In some examples, the first ABD includes 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.

[0239] In some examples, the first ABD includes 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.

[0240] In some examples, the first ABD includes 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.

[0241] In some examples, the first ABD includes 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.

[0242] In some examples, the first ABD includes 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.

[0243] In some examples, the first ABD includes 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.

[0244] In some examples, the first ABD includes 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.

[0245] In some examples, the first ABD includes 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.

[0246] In some examples, the first ABD includes 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.

[0247] In some examples, the first ABD includes 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.

[0248] In some examples, the first ABD includes 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.

[0249] In some examples, the first ABD includes 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.

[0250] In some examples, the first ABD includes 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.

[0251] In some examples, the first ABD includes 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.

[0252] In some examples, the first ABD includes 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.

[0253] In some examples, the first ABD includes 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.

[0254] In some examples, the first ABD includes 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.

[0255] In some examples, the first ABD includes 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.

[0256] In some examples, the first ABD includes 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.

[0257] In some examples, the first ABD includes 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.

[0258] In some examples, the first ABD includes 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.

[0259] In some examples, the first ABD includes 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.

[0260] In some examples, the first ABD includes 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.

[0261] In some examples, the first ABD includes 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.

[0262] In some examples, the first ABD includes 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.

[0263] In some examples, the first ABD includes 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.

[0264] In some examples, the first ABD includes 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.

[0265] In some examples, the first ABD includes 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.

[0266] In some examples, the first ABD includes 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.

[0267] In some examples, the first ABD includes 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.

[0268] In some examples, the first ABD includes 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.

[0269] In some examples, the first ABD includes 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.

[0270] In some examples, the first ABD includes 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.

[0271] In some examples, the first ABD includes 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.

[0272] In some examples, the first ABD includes 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.

[0273] In some examples, the first ABD includes 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.

[0274] In some examples, the first ABD includes 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.

[0275] In some examples, the first ABD includes 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.

[0276] In some examples, the first ABD includes 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.

[0277] In some examples, the first ABD includes 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.

[0278] In some examples, the first ABD includes 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.

[0279] In some examples, the first ABD includes 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.

[0280] In some examples, the first ABD includes 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.

[0281] In some examples, the first ABD includes 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.

[0282] In some examples, the first ABD includes 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.

[0283] In some examples, the first ABD includes 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.

[0284] In some examples, the first ABD includes 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.

[0285] In some examples, the first ABD includes 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.

[0286] In some examples, the first ABD includes 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.

[0287] In some examples, the first ABD includes 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.

[0288] In some examples, the first ABD includes 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.

[0289] In some examples, the first ABD includes 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.

[0290] In some examples, the first ABD includes 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.

[0291] In some examples, the first ABD includes 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.

[0292] In some examples, the first ABD includes 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.

[0293] In some examples, the first ABD includes 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.

[0294] In some examples, the first ABD includes 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.

[0295] In some examples, the first ABD includes 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.

[0296] In some examples, the first ABD includes 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.

[0297] In some examples, the first ABD includes 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.

[0298] In some examples, the first ABD includes 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.

[0299] In some examples, the first ABD includes 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.

[0300] In some examples, the first ABD includes 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.

[0301] In some examples, the first ABD includes 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.

[0302] In some examples, the first ABD includes 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.

[0303] In some examples, the first ABD includes 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.

[0304] In some examples, the first ABD includes 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.

[0305] In some examples, the first ABD includes 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.

[0306] In some examples, the first ABD includes 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.

[0307] In some examples, the first ABD includes 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.

[0308] In some examples, the first ABD includes 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.

[0309] In some examples, the first ABD includes 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.

[0310] In some examples, the first ABD includes 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.

[0311] In some examples, the first ABD includes 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.

[0312] In some examples, the first ABD includes 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.

[0313] In some examples, the first ABD includes 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.

[0314] In some examples, the first ABD includes 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.

[0315] In some examples, the first ABD includes 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.

[0316] In some examples, the first ABD includes 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.

[0317] In some examples, the first ABD includes 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.

[0318] In some examples, the first ABD includes 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.

[0319] In some examples, the first ABD includes 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.

[0320] In some examples, the first ABD includes 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.

[0321] In some examples, the first ABD includes 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.

[0322] In some examples, the first ABD includes 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.

[0323] In some examples, the first ABD includes 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.

[0324] In some examples, the first ABD includes 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.

[0325] In some examples, the first ABD includes 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.

[0326] In some examples, the first ABD includes 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.

[0327] In some instances, the first ABD includes the HCV domain of 24D9, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine. In some instances, the first ABD includes the LCV domain of 24D9, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine. In some instances, the first ABD includes the HCV domain of 24D9, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by histidine; and the LCV domain of 24D9, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted by 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.

[0328] 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 one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions in total substituted with histidine in common in SEQ ID NOs: 513-515. 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 one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions in total substituted with histidine in common in SEQ ID NOs: 519-521. In some examples, the first ABD includes: an HCV domain, which comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 513, 514 and 515, respectively, and optionally 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 are commonly substituted by histidine; and an LCV domain, which comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 519, 520 and 521, respectively, and optionally 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 are commonly substituted by histidine.

[0329] In some instances, the first ABD includes an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, optionally wherein the HCV domain includes 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 instances, the first ABD includes an LCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, optionally wherein the LCV domain includes 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 consisting of: 35 and 97. In some examples, the first ABD includes an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:512, optionally wherein the HCV domain includes 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; and an LCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, optionally wherein the LCV domain includes 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 SEQ ID NO:518 or SEQ ID NO:522 comprises 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.

[0330] In some instances, 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 shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCV domain includes 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.

[0331] Table 3. Exemplary combinations of amino acid positions in SEQ ID NO: 512 or SEQ ID NO: 516 that can be substituted with histidine

[0332]

[0333]

[0334] In some instances, the LCV domain includes an LCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCV domain includes 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.

[0335] Table 4. Exemplary combinations of amino acid positions in SEQ ID NO: 518 or SEQ ID NO: 522 that can be substituted with histidine

[0336]

[0337] In some examples, the first ABD comprises an HCV domain comprising a sequence of SEQ ID NO:512, SEQ ID NO:516, or one of SEQ ID NOs:523-571; and / or the first ABD includes an LCV domain comprising a sequence shown in SEQ ID NO:518 or SEQ ID NO:522.

[0338] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence shown in SEQ ID NO:512 or SEQ ID NO:516 or one of SEQ ID NOs:523-567.

[0339] In some examples, the first ABD includes an LCV domain comprising the sequence shown in SEQ ID NO:518 or SEQ ID NO:522 and an HCV domain comprising the sequence shown in SEQ ID NO:516 or one of SEQ ID NOs:568-571.

[0340] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:512.

[0341] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:516.

[0342] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:523.

[0343] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:524.

[0344] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:525.

[0345] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:526.

[0346] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:527.

[0347] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:528.

[0348] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:529.

[0349] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:530.

[0350] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:531.

[0351] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:532.

[0352] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:533.

[0353] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:534.

[0354] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:535.

[0355] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:536.

[0356] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:537.

[0357] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:538.

[0358] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:539.

[0359] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:540.

[0360] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:541.

[0361] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:542.

[0362] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:543.

[0363] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:544.

[0364] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:545.

[0365] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:546.

[0366] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:547.

[0367] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:548.

[0368] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:549.

[0369] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:550.

[0370] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:551.

[0371] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:552.

[0372] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:553.

[0373] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:554.

[0374] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:555.

[0375] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:556.

[0376] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:557.

[0377] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:558.

[0378] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:559.

[0379] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:560.

[0380] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:561.

[0381] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:562.

[0382] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:563.

[0383] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:564.

[0384] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:565.

[0385] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:566.

[0386] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises the sequence of SEQ ID NO:567.

[0387] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises SEQ ID NO:512.

[0388] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises SEQ ID NO:516.

[0389] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises SEQ ID NO:568.

[0390] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises SEQ ID NO:569.

[0391] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprises SEQ ID NO:570.

[0392] In some examples, the first ABD includes an LCV domain and a HCV domain, the LCV domain comprising the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, and the HCV domain comprising SEQ ID NO:571.

[0393] In some examples, the first ABD includes the 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.

[0394] In some examples, the first ABD includes the LCV domain of 29F1, optionally having 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.

[0395] In some examples, the first ABD includes the HCV domain of 29F1, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted with histidine, aspartic acid or glutamate; and the LCV domain of 29F1, which optionally has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids substituted with histidine, aspartic acid or glutamate.

[0396] In some examples, the HCV domain of 29F1 comprises SEQ ID NO:572 or SEQ ID NO:572.

[0397] In some examples, the LCV domain of 29F1 comprises SEQ ID NO:577 or SEQ ID NO:581.

[0398] 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 in which 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 are commonly substituted with histidine, aspartic acid or glutamic acid.

[0399] 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 in which 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 are commonly substituted with histidine, aspartic acid or glutamic acid.

[0400] In some examples, the first ABD includes: an HCV domain, wherein the HCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 154, 155 and 156, respectively, and optionally in SEQ ID NOs: 573-575, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions are commonly substituted by histidine, aspartic acid or glutamic acid; and an LCV domain, wherein the LCV domain comprises CDR1, CDR2 and CDR3 of SEQ ID NOs: 157, 158 and 159, respectively, and optionally in SEQ ID NOs: 578-580, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid positions are commonly substituted by histidine, aspartic acid or glutamic acid.

[0401] In some instances, the first ABD includes an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:572 or SEQ ID NO:576, optionally wherein the HCV domain includes 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 following group: a D position selected from 31, 56, and 99; and an E position selected from 59. In some examples, the first ABD includes an LCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, optionally wherein the LCV domain includes 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 following group: a D position selected from 27, 28, 31, 52 and 56; or an E position selected from 51 and 56. In some examples, the first ABD includes an HCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO: 572 or SEQ ID NO: 576, optionally wherein the HCV domain includes 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 consisting of: 27, 32, 33, 34, 35, 51, 54, 56, 58, and 100; and an LCV domain that is identical to the sequence shown in SEQ ID NO: 577 or SEQ ID NO: The sequence shown in NO:581 is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99% or 100%) identical, optionally wherein 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

[0402] In some instances, 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 shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCV domain includes 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.

[0403] Table 5. Exemplary combinations of amino acid positions in SEQ ID NO: 572 or SEQ ID NO: 576 that can be substituted with histidine, aspartic acid, or glutamic acid

[0404]

[0405] In some instances, the LCV domain includes an LCV domain that is at least 90% (e.g., ≥92%, ≥94%, ≥96%, ≥98%, ≥99%, or 100%) identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCV domain includes 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.

[0406] Table 6. Exemplary combinations of amino acid positions in SEQ ID NO: 577 or SEQ ID NO: 581 that can be substituted with histidine, aspartic acid, or glutamic acid

[0407]

[0408]

[0409] In some examples, the first ABD comprises an HCV domain comprising a 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 a 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.

[0410] In some examples, the first ABD comprises an LCV domain comprising the sequence in 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.

[0411] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:581, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 625, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 626, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 627, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 628, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain includes the sequence of SEQ ID NO: 629, and the HCV domain includes 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 630, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 631, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 632, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 633, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 634, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 635, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 636, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 637, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 638, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 639, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 640, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 641, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 642, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 643, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 644, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 645, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 646, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 647, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 648, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 649, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 650, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 651, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 652, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 696, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 697, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 698, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 699, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 700, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:701, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 702, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 703, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 704, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:705, and the HCV domain comprises 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 includes an LCV domain and a HCV domain, wherein the LCV domain includes the sequence of SEQ ID NO:706, and the HCV domain includes 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] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 707, and the HCV domain comprises 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.

[0452] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:708, and the HCV domain comprises 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.

[0453] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:709, and the HCV domain comprises 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.

[0454] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:710, and the HCV domain comprises 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.

[0455] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:711, and the HCV domain comprises 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.

[0456] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:712, and the HCV domain comprises 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.

[0457] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:713, and the HCV domain comprises 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.

[0458] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:714, and the HCV domain comprises 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.

[0459] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:715, and the HCV domain comprises 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.

[0460] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain includes the sequence of SEQ ID NO:716, and the HCV domain includes 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.

[0461] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:717, and the HCV domain comprises 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.

[0462] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:718, and the HCV domain comprises 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.

[0463] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:719, and the HCV domain comprises 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.

[0464] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:720, and the HCV domain comprises 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.

[0465] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:721, and the HCV domain comprises 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.

[0466] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO: 722, and the HCV domain comprises 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.

[0467] In some examples, the first ABD includes an LCV domain and a HCV domain, wherein the LCV domain comprises the sequence of SEQ ID NO:723, and the HCV domain comprises 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.

[0468] Also provided herein are pharmaceutical compositions comprising any of the ABPCs described herein.

[0469] Also provided herein are methods of treating a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any of the ABPCs described herein.

[0470] In some examples, a composition including 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 including the same amount of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., any of the percentage increases or ranges of percentage increases listed on pages 389-404 of WO 2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984, which is incorporated herein by reference in its entirety)).

[0471] In some examples, a composition including 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 including the same amount of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., any of the increase-folds or ranges of increase-folds listed on pages 404-420 of WO 2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984, which is incorporated herein by reference in its entirety)).

[0472] 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 target mammalian cell killing compared to a composition comprising the same amount of a control ABPC (e.g., an increase of ≥ 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%). %, 80%, 85%, 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 1% to 10,000%).

[0473] In some embodiments, a composition comprising an ABPC (e.g., any of the ABPCs described herein) can provide an increase (e.g., a detectable increase) in target mammalian cell killing compared to a composition comprising the same amount of a control ABPC (e.g., an increase of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 times, or at least 100 times, or 0.1-100 times).

[0474] In some examples, the IC of the composition comprising the same amount of control ABPC is 50 In comparison, the IC of a composition comprising any of the ABPCs described herein (eg, when contacted with a target mammalian cell presenting LRRC15 on its surface) 50 (for target mammalian cell killing) reduction (e.g., a reduction of ≥1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or a reduction of at least 99%, a reduction of about 1%-99%, or any subrange of this range described herein).

[0475] In some examples, for example, a composition comprising any of the ABPCs described herein (e.g., when contacted with a target mammalian cell presenting LRRC15 on its surface) can provide a K on a target mammalian cell presenting LRRC15 on its surface at neutral pH, for example, compared to a control ABPC. D Compared with the IC on the same target cells at neutral pH 50of the invention (e.g., by at least 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 times, or by at least 100 times, or by about 0.1-500 times (or any subrange of this range described herein)). 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 endolysosomal delivery in target mammalian cells compared to a composition comprising the same amount of a control ABPC (e.g., at least an increase of 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 200%, 5%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1,000%, 2,000%, 3,000%, 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, or at least 10,000%, or an increase of about 1%-10,000% (e.g., or any of the subranges of this range described herein)). In some examples, a composition comprising ABPCs can provide an increase (e.g., an increase of ≥ 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, 3.6, 3.7, 3.8, 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, 7.9, 8.10, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.8, 8.9, 9.10, 9.11, 9.12, 9.13, 9.14, 9.15 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, or at least 100 fold, or about 0.1-100 fold).In any of the examples of ABPCs described herein, the target mammalian cell does not express an FcRn receptor, or expresses a lower (e.g., detectably lower) level (e.g., at least 1% lower, at least 2%, 5%, 10% lower, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% lower) level of an FcRn receptor compared to a control cell expressing FcRn (e.g., HUVEC—ThermoFisher No. C0035C). In some examples, the target mammalian cell is a cancer cell. In some examples, the ABPC is cytotoxic or cytostatic to the target mammalian cell. In some examples, a composition comprising any of the ABPCs described herein (e.g., when administered to a subject) causes a reduction in the level of LRRC15 presented on the surface of a target cell that is less (e.g., a 1% reduction to about 99% reduction or any sub-range of this range described herein) than 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 cause a detectable reduction in the level of LRRC15 presented on the surface of a target mammalian cell. In some examples, the ABPCs are cross-reactive with non-human primate LRRC15 and human LRRC15. In some examples, the ABPCs are cross-reactive with one or both of non-human primate LRRC15, human LRRC15, and rat LRRC15 and mouse LRRC15. In some examples, the ABPCs are cross-reactive with non-human primate LRRC15, human LRRC15, rat LRRC15, and mouse LRRC15. In some instances, the ABPCs are cross-reactive with mouse LRRC15 and rat LRRC15. In some instances, the ABDs bind 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 aspects of these methods are described below and may be used in any combination without limitation. Additional aspects of these methods are known in the art.

[0476] LRRC15 or epitope of LRRC15

[0477] A variety of LRRC15 binding monoclonal antibodies have been described in the literature, which 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.

[0478] Antigen binding protein constructs

[0479] Any of the antigen binding protein constructs (ABPCs) described herein may be a single polypeptide, or may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 (same or different) polypeptides. In some embodiments where the ABPC is a single polypeptide, the ABPC may include a single ABD or two ABDs. In some embodiments where the ABPC is a single polypeptide and includes two ABDs, the first ABD and the second ABD may be the same or different from each other (and may specifically bind to the same or different antigens or epitopes).

[0480] In some embodiments where the ABPC is a single polypeptide, the first ABD and the second ABD (if present) can each be independently selected from the group consisting of: a VH domain, a VHH domain, a VNAR domain, and a 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 bifunctional antibody, sc bifunctional antibody-CH3, scFv-CH-CL-scFv, HSAbody, sc bifunctional antibody-HAS, tandem-scFv, Adnectin, DARPin, fibronectin, DEP conjugate, PROTAB and PROTAC.

[0481] In some embodiments, the ABPC or the ABD comprises a proteolysis targeting antibody (PROTAB) (as described, for example, in Marei et al., "Antibody targeting of E3 ubiquitin ligases for receptor degradation". Nature, October 6, 2022). In some embodiments, PROTABs or PROTACs can link cell surface E3 ubiquitin ligases to transmembrane proteins, causing target degradation both in vitro and in vivo.

[0482] Additional examples of ABDs that can be used when the ABPC is a single polypeptide are known in the art.

[0483] 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. HH domain and V NAR Non-limiting aspects 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 in Biotechnology 30:187-198, 2006; 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 in Pharmacological Sciences 38:21-28, 2016; 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., E Biomedicine 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.

[0484] In some embodiments where the ABPC is a single polypeptide and includes two ABDs, both the first ABD and the second ABD can be VHH domains, or at least one ABD can be a VHH domain. NAR domain, or at least one ABD is V NAR In some embodiments where the ABPC is a single polypeptide, the first ABD is a scFv domain. In some embodiments where the ABPC is a single polypeptide and includes two ABDs, both the first ABD and the second ABD can be scFv domains, or at least one ABD can be a scFv domain.

[0485] In some embodiments, the ABPC may include two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides). In some embodiments in which the ABPC includes two or more polypeptides, two, three, four, five, or six of the two or more polypeptides may be identical.

[0486] In some embodiments where the ABPC comprises two or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides), the two or more polypeptides of the ABPC can be assembled (e.g., non-covalently assembled) 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 bi-scFab, a F(ab')2, a diabody, a crossMab, a DAF (two-in-one) 、DAF (four-in-one), DutaMab, DT-IgG, common light chain of knob and hole, knob and 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, Triabody, Minibody, Minibody, TriBi Microbody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scdiabody-Fc, Diabody-Fc, Tandem scFv-Fc, VHH-Fc, Tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, Dock-Lock Antibody, ImmTAC, IgG-IgG Conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, Fibronectin, and DEP Conjugate. 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.

[0487] Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab') 2Fragments and Fab' fragments. Additional examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., human or humanized IgG, such as antigen-binding fragments of human or humanized IgG1, IgG2, IgG3, or IgG4); antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA, such as antigen-binding fragments of 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).

[0488] The "Fv" fragment comprises a non-covalently linked dimer of one HCV domain and one LCV domain.

[0489] In addition to the heavy and LCV domains of the Fv fragment, the "Fab" fragment also includes the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ).

[0490] “F(ab') 2 The " fragment consists of two Fab fragments linked by a disulfide bond near the hinge region.

[0491] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins as described, for example, in DiGiammarino et al., Methods Mol. Biol. 899: 145-156, 2012; Jakob et al., MABs 5: 358-363, 2013; and in U.S. Pat. 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.

[0492] DARTs are described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0493] Additional aspects of ABPCs are known in the art.

[0494] Antigen binding domain

[0495] In some embodiments of any of the antigen binding protein constructs (ABPCs) described herein, the first ABD (and optionally the second ABD, if present) is at a pH of about 4.0 to about 6.5 (e.g., about 4.0 to about 6.4, or any of the pH ranges listed on pages 428-430 of WO2021 / 022039 (or corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference. The off-rate ratio at a pH of about 7.0 to about 8.0 (e.g., about 7.0-7.9, 7.0-7.8, 7.0-7.7, 7.0-7.6, 7.0-7.5, 7.0-7.4, 7.0-7.3, 7.0-7.2, 7.0-7.1, 7.1-8.0, 7.1-7.9, 7.1-7.8, 7.1-7.7, 7.1-7.6, 7.1-7.5, 7.1-7.4, 7.0-7.3, 7.0-7.2, 7.0-7.1, 7.1-8.0, 7.1-7.9, 7.1-7.8, 7.1-7.7, 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, 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) under a fast off rate (for example, at least 5% faster, or the faster % or % range listed on pages 430-447 of WO 2021 / 022039 (or in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entirety).

[0496] 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 of this range described herein) is 0.0045 for the first ABD (and optionally the second ABD, if present) and 0.0066 for the second ABD ( D ) is greater than the K at a pH of about 7.0 to about 8.0 (e.g., any of the subranges of this range described herein). D3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or at least about 10,000% greater than, or about 5% to about 10,000% greater than, or about 15% to about 20,000% greater than, or about 25% to about 30,000% greater than, or about 5% to about 10,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, or about 15% to about 20,000% greater than, K listed on pages 448-466 of 2021 / 022039 (or listed in corresponding U.S. patent application No. 2022 / 0281984) D any of the larger percentage ranges of , which is incorporated herein by reference in its entirety).

[0497] 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 sub-ranges of this range described herein) is faster (e.g., at least 0.2 times faster, or any of the faster times or ranges of faster times listed in WO 2021 / 022039, pp. 466-468 (or in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entireties) than the dissociation rate at a pH of about 7.0 to about 8.0 (e.g., or any of the sub-ranges of this range described herein).

[0498] 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 of this range described herein) is 0.0045 for the first ABD (and optionally the second ABD, if present) and 0.0066 for the second ABD ( D ) is greater than (e.g., detectably greater than) 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 (e.g., at least 0.2 times greater, or any of a greater multiple or range of greater multiples listed on pages 468-470 of WO 2021 / 022039 (or listed in corresponding U.S. Patent Application No. 2022 / 0281984), which are incorporated herein by reference in their entirety).

[0499] In some embodiments of the ABPC comprising a first ABD and a second ABD, the first ABD and the second ABD are identical or at least 80% identical to each other in amino acid sequence (e.g., ≥82%, ≥84%, ≥86%, ≥88%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identical). In some embodiments of the ABPC comprising a first ABD and a second ABD, the sequences of the first ABD and the second ABD are less than 80% identical (e.g., <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, or <5% identical). In some embodiments of the ABPC comprising a first ABD and a second ABD, the first ABD and the 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).

[0500] In some embodiments of any of the ABPCs described herein, the first ABD (and optionally the second ABD, if present) has a K of about 1.0-1.0 at a pH of about 7.0-8.0 (e.g., any of the subranges of this range described herein). D The K is about 1 pM-5 μM (e.g., 1 pM-2 μM, or as listed on pages 471-492 of WO 2021 / 022039 (or as listed in the corresponding U.S. Patent Application No. 2022 / 0281984) D , which is incorporated herein by reference in its entirety).

[0501] In some embodiments of any of the ABPCs described herein, the first ABD (and optionally the second ABD, if present) has a K of about 1.0-1.0 at a pH of about 4.0-6.5 (e.g., any of the subranges of this range described herein). D The K may be greater than 1 nM (e.g., about 1 nM-1 mM, or the K values ​​listed on pages 492-496 of WO 2021 / 022039 (or the K values ​​listed in the corresponding U.S. Patent Application No. 2022 / 0281984) D , which is incorporated herein by reference in its entirety).

[0502] A variety of different methods known in the art can be used to determine the K of any of the antigen binding protein constructs described herein. Dvalues ​​(e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, biomolecule binding kinetics assays, in vitro binding assays on antigen-expressing cells, etc.).

[0503] In some examples, the in vivo half-life of the ABPC is reduced (e.g., detectably reduced) compared to the half-life of a control ABPC (e.g., any of the exemplary control ABPCs described herein) (e.g., at least 1%, or a percentage reduction or range of percentage reductions listed on pages 496-500 of WO 2021 / 022039 (or corresponding U.S. Patent Application No. 2022 / 0281984, which are incorporated herein by reference in their entireties). Conversely, in some examples, the in vivo half-life of the ABPC may be increased compared to the half-life of the control ABPC.

[0504] Conjugation

[0505] In some embodiments, the ABPCs provided herein can be conjugated to drugs (e.g., chemotherapeutic drugs, small molecules), toxins, or radioisotopes. Non-limiting examples of drugs, toxins, and radioisotopes are known in the art.

[0506] In some embodiments, at least one polypeptide of any ABPC 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 can be cleaved on the ABPC once transported to a lysosome or late endosome by a target mammalian cell. In some embodiments, cleavage of the linker functionally activates the drug or toxin.

[0507] In some embodiments, at least one polypeptide of any ABPC described herein is conjugated to a toxin, a radioisotope, or a drug via a non-cleavable linker. In some embodiments, the conjugated toxin, radioisotope, or drug is released during lysosomal and / or late endosomal degradation of the ABPC.

[0508] 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 Part 1):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.

[0509] Non-limiting examples of non-cleavable linkers include: maleimidoalkane linkers and maleimidocyclohexane linkers (MMC) (see, e.g., those described in McCombs et al., Journal of the American Association of Pharmaceutical Scientists (AAPS J.) 17(2):339-351, 2015).

[0510] In some embodiments, any of the ABPCs described herein are cytotoxic or cytostatic to target mammalian cells.

[0511] In some embodiments, antibodies provided herein may include one or more amino acid substitutions to provide a conjugation site (e.g., conjugated to a drug, toxin, radioisotope). In some embodiments, antibodies provided herein may have one conjugation site. In some embodiments, antibodies described herein may have two conjugation sites. In some embodiments, antibodies provided herein may have three or more conjugation sites. Non-limiting examples of amino acid substitutions that produce a conjugation site (e.g., a "three-hinge" conjugation site) are described in U.S. Patent Application No. 2017 / 0348429, which is incorporated herein by reference in its entirety. For example, lysine at amino acid position 105 of SEQ ID NO:351 or SEQ ID NO:352 becomes a substitution of cysteine ​​and threonine at amino acid positions 106 and 108 can provide a "three-hinge" conjugation site in any one of the antibodies described herein. In some embodiments, a substitution of alanine at amino acid position 1 of SEQ ID NO:351 or 352 becomes a substitution of cysteine ​​that can provide a conjugation site for any one of the antibodies described herein. In some embodiments, a substitution of Val to Cys at position 98 of SEQ ID NO: 353 can provide a conjugation site for any of the antibodies described herein.

[0512] Naturally occurring cysteine ​​amino acids can also provide conjugation (e.g., conjugation with drugs, toxins, radioisotopes). In some embodiments, the antibodies provided herein can have drugs, toxins, or radioisotopes conjugated at one or more (e.g., one, two, three, or four) naturally occurring conjugation sites. In some embodiments, the cysteine ​​at amino acid position 103 of SEQ ID NO: 351 or 352 is a naturally occurring conjugation site. In some embodiments, the cysteine ​​at amino acid position 109 of SEQ ID NO: 351 or 352 is a naturally occurring conjugation site. In some embodiments, the cysteine ​​at amino acid position 112 of SEQ ID NO: 5 or 6 is a naturally occurring conjugation site. In some embodiments, the cysteine ​​at amino acid position 107 of SEQ ID NO: 353 is a naturally occurring conjugation site.

[0513] 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) naturally occurring conjugation sites (e.g., cysteine ​​at amino acid position 103 of SEQ ID NO: 351 or 352, cysteine ​​at amino acid position 109, and / or cysteine ​​at amino acid position 112, and / or cysteine ​​at amino acid position 107 of SEQ ID NO: 353). In some embodiments, the antibodies provided herein can have a drug, toxin, or radioisotope conjugated at one or more naturally occurring conjugation sites and one or more engineered conjugation sites.

[0514] The conjugation by engineered cysteine ​​is achieved by methods known in the art. In short, antibodies containing engineered cysteine ​​are prepared by treatment with a reducing agent, for example, tris (2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT) or 2-mercaptoethanol (BME) for conjugation. In the reduction reaction, the reducing agent is combined with the disulfide bonds in the antibody, destroying the interchain disulfide and removing the disulfide cap from the engineered cysteine. The optional reoxidation step realized by exposing the solution to air or an oxidant (such as dehydroascorbic acid) allows the reorganization of the interchain disulfide bond, so that the engineered cysteine ​​has a thiolate reactive group. The conjugation with a functional group on the joint payload, i.e. maleimide-vc-MMAE, is achieved by reacting with the payload in a buffer solution containing cosolvents such as ethanol, dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO). By size exclusion chromatography or selective filtration methods, such as tangential flow filtration, the crude conjugated antibody solution is purified. In this step, residual unreacted payload, reducing agent, and oxidizing agent are removed from the reaction mixture, and the conjugated ADC product can be transferred into the desired formulation buffer.

[0515] By a similar method, antibodies with or without additional engineered cysteine ​​conjugation sites are used to achieve conjugation through hinge cysteine. In short, antibodies are prepared by treating with a reducing agent, for example, tris (2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT) to carry out conjugation. The reducing strength and concentration of the reducing agent are selected so that some or all of the interchain disulfide bonds in the interchain disulfide bonds are reduced, leaving free cysteine ​​for conjugation. The solution can be directly conjugated in the presence of an excess of a reducing agent. The conjugation with a functional group on a joint payload, i.e. maleimide-vc-MMAE, is achieved by reacting with a payload in a buffer solution containing cosolvents such as ethanol, dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO). Unreacted joint payloads can become unreactive by adding sacrificial thiolate molecules (such as acetylcysteine). 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. Separation of the chromatographic fractions allows selection of the desired antibody to payload ratio and removal of unreacted antibody, protein aggregates and fragments, and payload-related reaction byproducts. The purified antibody drug conjugate can be further purified by size exclusion chromatography or selective filtration methods such as TFF. In this step, the conjugated ADC product can also be transferred to the desired formulation buffer.

[0516] In some examples, an antibody conjugate comprising an antibody linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereinafter referred to as LRRC15-IgG-DC) can be prepared. Conjugation of the antibody to vcMMAE begins with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (10 mg / mL) was partially reduced by the addition of TCEP (2:1 molar equivalents of TCEP:mAb) followed by overnight incubation at 4°C. The reduction reaction was then warmed to 25°C. To conjugate all thiols, vcMMAE was added to a final vcMMAE:reduced Cys molar ratio of 1:10. The conjugation reaction was performed in the presence of 10% v / v dimethylacetamide (DMA) and allowed to proceed at 25°C for 60 minutes.

[0517] In some examples, an antibody conjugate (ADC) was prepared comprising a LRRC15 binding IgG described herein (hereinafter referred to as LRRC15-IgG) linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereinafter referred to as LRRC15-IgG-DC). Conjugation of the antibody to vcMMAE began with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (10 mg / mL) was reduced by addition of DTT (molar equivalent of DTT:mAb was 100:1) followed by overnight incubation at 25°C. The reduced LRRC15-IgG (10 mg / mL) was then reoxidized by exposure to DHAA (molar equivalent of DHAA:mAb was 10:1) followed by incubation at 25°C for 2 hours. To conjugate all thiols, vcMMAE was added to achieve a final vcMMAE:mAb molar ratio of 4: 1. The conjugation reaction was performed in the presence of 10% v / v DMA and allowed to proceed at 25°C for 3 hours.

[0518] Expression of antigen binding protein constructs in cells

[0519] Also provided herein is a method for generating a recombinant cell expressing an ABPC (e.g., any of the ABPCs described herein), the method comprising: introducing a nucleic acid encoding the ABPC into a cell to produce a recombinant cell; and culturing the recombinant cell under conditions sufficient to express the ABPC. In some embodiments, the introducing step comprises introducing an expression vector comprising a nucleic acid encoding the ABPC into the cell to produce a recombinant cell.

[0520] Any ABPC described herein can be produced by any cell, such as a eukaryotic cell or a prokaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a nucleus bound to different membranes. Such cells may 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, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant or insect cell. As used herein, the term "prokaryotic cell" refers to a cell having a nucleus not bound to different membranes. In some embodiments, the prokaryotic cell is a bacterial cell.

[0521] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that are conducive to proliferation, differentiation and growth. In short, cells can be cultured by contacting cells (e.g., any cells) with a cell culture medium that includes essential growth factors and supplements that support cell viability and growth.

[0522] 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, piercing infection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

[0523] The methods provided herein further include isolating ABPCs 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).

[0524] Affinity

[0525] Antibodies and their antigen-binding fragments are multivalent, and therefore comprise more than one binding site. Typically, the measurement of the total binding strength of an antibody at its binding site is referred to as avidity. Generally, the term "avidity multiple" and "selectivity" may refer to the difference multiple between the affinity of an antibody and the avidity of an antibody, for example, when measuring the total binding strength of an antibody on a cell line with high target expression (avidity; For example, cancer cells, for example, SAOS-2 cells) compared with the total binding strength of an antibody on a cell line with low target expression (affinity, for example, non-cancerous cells, for example, G-292 cells). Typically, avidity is determined by four factors: binding affinity (for example, the strength of binding at a single binding site); valence (for example, the total number of binding sites); structural arrangement (for example, the structure of an antigen and an antibody); and antigen density (for example, the number of antigens per cell).

[0526] Provided herein are methods of reducing the risk of metastasis in a subject having cancer, or reducing the risk of the subject developing additional metastases, wherein the cancer is characterized by having a population of cancer cells having LRRC15 or an epitope of LRRC15 presented on the surface of the cancer cells, the method comprising administering to a subject identified as having the cancer a therapeutically effective amount of any of the antibodies described herein or any of the pharmaceutical compositions described herein. In some embodiments, the selectivity of an antibody described herein for cancer cells as compared to non-cancerous cells may be increased by 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. %, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%, 305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 395% or 400%. In some embodiments, the cancer is a sarcoma, for example, a leiomyosarcoma, an osteosarcoma, or a chondrosarcoma.

[0527] Treatment

[0528] Provided herein is a method for treating cancer characterized by having a population of cancer cells having LRRC15 or an epitope of LRRC15 presented on the surface of the cancer cells, the method comprising: administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having cancer, the cancer characterized by having a population of the cancer cells.

[0529] Also provided herein is a method of reducing the volume of a tumor in a subject, wherein the tumor is characterized by having a population of cancer cells having LRRC15 or an epitope of LRRC15 presented on the surface of the cancer cells, the method comprising: administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having cancer, wherein the cancer is characterized by having a population of the cancer cells. In some embodiments of any of the methods described herein, the volume of at least one cancer or tumor location 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 at least one tumor prior to administration of the ABPCs.

[0530] Also provided herein is a method of inducing cell death in a cancer cell in a subject, wherein the cancer cell has LRRC15 or an epitope of LRRC15 presented on the surface of the cancer cell, the method comprising: administering a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein to a subject identified as having cancer, wherein the cancer is characterized by a population of the cancer cells. In some embodiments, the induced cell death is necrosis or apoptosis.

[0531] 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 infiltrated tumor. In some embodiments of any of the methods described herein, the cancer is a T cell infiltrated 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, e.g., a leiomyosarcoma, an osteosarcoma, or a chondrosarcoma.

[0532] Provided herein are methods of reducing the risk of metastasis in a subject having cancer or reducing the risk of additional metastasis in the subject, wherein the cancer is characterized by having a population of cancer cells having LRRC15 or an epitope of LRRC15 presented on the surface of the cancer cells, the method comprising: administering to a subject identified as having cancer a therapeutically effective amount of any of the pharmaceutical compositions described herein or any of the ABPCs described herein. In some embodiments, a subject's risk of developing metastasis, or the risk of developing additional metastases, is reduced by ≥ 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 who is not administered treatment or treatment that does not include administration of any of the ABPCs described herein.

[0533] The term "subject" refers to any mammal. In some embodiments, the subject or "subject suitable for treatment" can be a canine (e.g., dog), a feline (e.g., cat), an equine (e.g., horse), an ovine, a bovine, a porcine, a caprine, a primate, such as a simian (e.g., a monkey (e.g., marmoset, baboon) or an ape (e.g., gorilla, chimpanzee, orangutan, or gibbon) or a 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, particularly a mammal that can be used routinely as a model for demonstrating therapeutic efficacy in humans (e.g., murine, lagomorph, porcine, canine, or primate).

[0534] As used herein, treatment 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 cancer described herein). For example, treatment can reduce cancer progression, reduce the severity of cancer, or reduce the risk of cancer recurrence in a subject with cancer.

[0535] Provided herein are methods of inhibiting the growth of a solid tumor in a subject (e.g., any subject described herein), comprising administering to the subject a therapeutically effective amount of any ABPC described herein or any pharmaceutical composition described herein (e.g., as compared to the growth of the solid tumor in the subject prior to treatment or the growth of a similar solid tumor in a different subject receiving a different treatment or not receiving treatment).

[0536] In some embodiments of any of the methods described herein, the growth of a solid tumor is the primary growth of a solid tumor. In some embodiments of any of the methods described herein, the growth of a solid tumor is the recurrent growth of a solid tumor. In some embodiments of any of the methods described herein, the growth of a solid tumor is the metastatic growth of a solid tumor. In some embodiments, the treatment causes the growth of a solid tumor in a subject (e.g., compared to the growth of a solid tumor in a subject before treatment or a growth of a similar solid tumor in a different subject receiving a different treatment or not receiving treatment) to decrease by about 1% to about 99% (or any sub-range of the sub-ranges of this range described herein). The growth of a solid tumor in a subject can be assessed by a variety of different imaging methods, such as positron emission tomography, X-ray computed tomography, computer axial tomography, and magnetic resonance imaging.

[0537] Also provided herein are methods of reducing the risk of metastasis or the risk of additional metastasis within a certain period of time in a subject identified as having a cancer (e.g., any exemplary cancer described herein), the method comprising administering to the subject a therapeutically effective amount of any protein described herein or any pharmaceutical composition described herein (e.g., compared to a subject having a similar cancer and receiving a different treatment or not receiving treatment). In some embodiments of any of the methods described herein, the metastasis or additional metastasis is to one or more of bone, lymph node, brain, lung, liver, skin, chest wall (including bone, cartilage and soft tissue), abdominal cavity, contralateral breast, soft tissue, muscle, bone marrow, ovary, adrenal gland and pancreas.

[0538] In some embodiments of any of the methods described herein, the time period 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, 4-1 0, 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).

[0539] In some embodiments, the risk of developing a metastasis or developing additional metastases over a period of time in a subject identified as having a cancer is reduced by about 1% to about 99% (e.g., or any of the subranges of this range described herein), e.g., compared to the risk in subjects with similar cancers, receiving different treatments, or not receiving treatment.

[0540] Non-limiting examples of cancer include: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary cause, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing's sarcoma, sarcoma), eye cancer, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi 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, occult primary metastatic squamous neck cancer, midline airway cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / bone marrow Hyperplastic tumors, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, 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, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms' tumor. Additional examples of cancer are known in the art.

[0541] In some embodiments, 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) are further administered to the patient. In some embodiments, the one or more additional therapeutic agents are administered to the patient at approximately the same time as any ABPC described herein is administered to the patient. In some embodiments, the one or more additional therapeutic agents are administered to the patient after any ABPC described herein is administered to the patient. In some embodiments, the one or more additional therapeutic agents are administered to the patient before any ABPC described herein is administered to the patient.

[0542] 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, e.g., leiomyosarcoma, osteosarcoma, or chondrosarcoma.

[0543] Composition

[0544] Also provided herein are compositions (eg, pharmaceutical compositions) comprising at least one of any of the ABPCs described herein. In some embodiments, the composition (eg, pharmaceutical composition) can be placed in a sterile vial or prefilled syringe.

[0545] In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular or intratumoral). In some embodiments, the composition (e.g., pharmaceutical composition) may include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline). Single or multiple administrations of any pharmaceutical composition described herein may be administered to a subject according to, for example, the dose 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 improve the condition, disease or symptom.

[0546] Also provided herein are methods of treating a subject having cancer (eg, any cancer described herein), the methods comprising administering a therapeutically effective amount of at least one of any of the compositions or pharmaceutical compositions provided herein.

[0547] Reagent test kit

[0548] Also provided herein are kits comprising any ABPC described herein, any composition described herein, or any pharmaceutical composition described herein. In some embodiments, the kit may include instructions for performing any method described herein. In some embodiments, the kit may include at least one dose of any composition described herein (e.g., pharmaceutical composition). In some embodiments, the kit may provide a syringe for administering any pharmaceutical composition as described herein.

[0549] Protein constructs

[0550] Also provided is a protein construct (PC), the PC comprising: a first ABD, the first ABD being capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell, wherein: (a) the first ABD has a faster dissociation rate at a pH of about 7.0 to about 8.0 (or any sub-range of the sub-ranges of this range described herein) than at a pH of about 4.0 to about 6.5 (or any sub-range of the sub-ranges of this range described herein); and / or (b) the first ABD has a dissociation constant (K) of about 7.0 to about 8.0 (or any sub-range of the sub-ranges of this range) of about 1. D ) is greater than the K at a pH of about 4.0 to about 6.5 D .

[0551] Also provided herein are pharmaceutical compositions comprising any PC described herein. Also provided herein are methods of treating a subject in need thereof comprising administering a therapeutically effective amount of any PC described herein.

[0552] Methods for improving pH dependence of antigen binding protein constructs

[0553] Also provided herein is a method for improving the pH dependence 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 the generation of an antigen-binding protein construct having one or both of the following: (a) an increase in the 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., an increase of at least 0.1-fold to an increase of about 100-fold, or any subrange of this range described herein), and (b) a dissociation constant (K) of the ABD at a pH of about 4.0 to about 6.5 compared to the starting antigen-binding protein construct. D ) and K at a pH of about 7.0 to about 8.0 DThe ratio of is increased (e.g., by at least 0.1 times to about 100 times, or any sub-range of the sub-ranges of this range described herein).

[0554] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0555] Examples

[0556] Example 1. Generation of LRRC15 binding factors and engineering of pH binding dependence

[0557] pH-engineered ABPCs specific for LRRC15 were generated using a variety of approaches. In the first approach, published or newly identified monoclonal antibodies against LRRC15 were used as starting templates for the introduction of mutations that allowed for the engineering of pH-dependent binding to LRRC15 and i) enhanced endolysosomal accumulation of conjugated toxins, and ii) enhanced recycling of LRRC15 to the cell surface. The second approach involved the discovery of newly generated ABPCs specific for LRRC15 from natural libraries or libraries with defined CDR composition by antibody display methods, and screening under conditions designed to select pH-engineered ABPCs specific for LRRC15. In each case, histidine residues play an important role in engineering pH-dependent binding proteins.

[0558] Since the pKa of histidine residues is 6.0, histidine residues are at least partially protonated at a pH below 6.5. Therefore, if the histidine side chain in the antigen binding domain participates in electrostatic binding interactions with its antigen, it will begin to become positively charged at a pH equal to or below 6.5. Based on the corresponding charge of the antigenic epitope and the interaction with the antigenic epitope, this can weaken or enhance the binding affinity of the interaction at a pH below 6.5. Therefore, the systematic introduction of histidine into the complementary determining region (CDR) of antibodies or other binding factor libraries (e.g., scFv libraries) can be used to identify substitutions that will affect the interaction of the antigen binding domain with the antigen at lower pH values. Therefore, the first method involves histidine scanning of the variable domain sequence of the disclosed monoclonal antibody to identify pH-dependent variants.

[0559] A variety of 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 in 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 fell into either or both of the Kabat and IMGT CDR definitions were referred to as CDR residues. To engineer pH-dependent sequence variants, individual amino acid residues within the heavy and / or light chain CDRs were systematically substituted with histidine, one at a time. Where the starting CDR residue was histidine, the histidine was mutated to alanine. Using methods known in the art, antibody variants with only one histidine or alanine mutation in the heavy chain / light chain CDRs are generated by co-transfecting Expi293 cells with a) one heavy chain or light chain sequence variant, and b) the corresponding starting ABPC (e.g., starting LRRC15 binding monoclonal antibody) light chain or heavy chain.

[0560] After allowing the protein expression period, the cell culture supernatant was collected, quantified, and the pH dependence of the variant was evaluated using biolayer interferometry (BLI) or other methods known in the art. In brief, the cell culture supernatant was normalized to an antibody expression level of 50 μg / mL and captured on an anti-human Fc sensor (Forte Biosciences). A baseline was established using 1× kinetic buffer (Forte Biosciences), and the sensor was associated with 100 nM LRRC15 in 1× PBS at pH 7.4 for 300 seconds to generate an association curve. In the dissociation phase, the antibody-antigen complex on the sensor was exposed to 1× PBS at pH 5.5 or pH 7.4 for 300-500 seconds. The association and dissociation phase curves at pH 5.5 and pH 7.4 for the starting ABPC antibody and each corresponding antibody variant were examined to provide information based on two criteria: a) enhanced dissociation at pH 5.5 due to histidine or alanine substitutions compared to the starting ABPC (i.e., higher koff values); and b) reduced dissociation at pH 7.4 compared to the antibody variant itself and the starting ABPC at pH 5.5 (i.e., lower koff values). Variants that showed enhanced dissociation at pH 5.5 or reduced dissociation at pH 7.4, or both, were selected for further analysis. It was also noted that while some histidine and alanine mutations abolished LRRC15 binding, other mutations were tolerated with little change (e.g., less than 1-fold change in KD or dissociation rate) or no change in LRRC15 binding kinetics. Particularly because histidine is a large, positively charged amino acid, these unchanged histidine variants and alanine variants are known for being positions that can tolerate extensive mutations and produce antibodies with different sequences but similar binding properties, which is an otherwise non-obvious name. Variants selected for further analysis were expressed on a larger scale and purified using protein A affinity chromatography. The binding kinetics (kon and koff) of purified starting ABPC and variant antibodies were measured using Biacore (GE healthcare) at pH 5.5 and pH 7.4. The ratio of antibody dissociation rates (koff at pH 7.4 divided by koff at pH 5.5) was also used as a quantitative assessment of pH-dependent binding; similarly, the dissociation constant KD was calculated as koff divided by kon at 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) was also used as a quantitative assessment of pH-dependent binding.

[0561] Antibodies with an off-rate less than the off-rate of the starting ABPC and / or a dissociation constant ratio less than the dissociation constant ratio of the starting ABPC are selected for further evaluation of the combination substitution. Favorable histidine and / or alanine amino acid positions can also be combined to enhance pH dependence; this can be accomplished by, for example, combinatorially or rationally combining histidine and / or alanine substitutions that improve pH dependence individually on a given heavy or light chain, for example, by combinatorially or rationally combining modified heavy and light chains so that histidine and / or alanine substitutions are present on both chains, or a combination thereof. Using the methods and protocols described herein or other methods and protocols known in the art, the differential pH dependence of such combination variants is generated and tested / analyzed. Antibody variants with the lowest off-rate ratio and / or dissociation constant ratio are selected as candidates for further analysis (hereinafter referred to as "ABPCs engineered with pH specificity for LRRC15").

[0562] The second method for selecting pH-engineered ABPCs specific for LRRC15 involves screening libraries to identify de novo pH-dependent ABPCs specific for LRRC15 or ABPCs that can be used 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 other libraries known in the art) or phage / yeast display libraries in which the CDRs have been mutated to express a subset of amino acid residues. Using methods known in the art, the library is screened against a soluble recombinant LRRC15 extracellular domain, and variants that bind weakly (e.g., eluted from beads) at pH 5.0 and strongly (e.g., bound to beads) at pH 7.4 are positively selected. Three rounds of selection are performed. The final round of binding factors are screened for binding to human LRRC15 as well as cynomolgus monkey LRRC15 and mouse LRRC15 using ELISA or by mean fluorescence intensity in flow cytometric analysis. If more binders with cynomolgus or murine cross-reactivity are desired, a final round of selection can be performed on cynomolgus LRRC15 or murine LRRC15 instead. Selected binding proteins are subcloned into mammalian expression vectors and expressed as complete IgG proteins or Fc fusions in Expi293 cells. BLI analysis is performed as described herein to select pH-dependent binding factor variants and confirmed using Biacore.

[0563] Generation of 15G7, 24D9, and 29F1 anti-LRRC15 monoclonal antibodies

[0564] The applicant inoculated rabbits with the extracellular domain of the human LRRC15 protein to produce 250 rabbit mAbs (RabMAbs). After the initial generation of b-cell clones, the cell supernatant was evaluated in a secondary cytotoxicity assay. Each Ab that killed the cells was sequenced, obtained as a chimeric antibody (i.e., a rabbit variable domain with a human constant region), expressed, and then re-assayed for cytotoxicity, internalization, and other properties. The top candidates were humanized, and at the same time, histidine, aspartic acid, glutamic acid, and other amino acid scanning variants of the chimeric antibodies were generated as described in U.S. Patent Application No. US2022 / 0281984, which is incorporated herein by reference in its entirety.

[0565] Once promising RabMAb mutations and humanized frameworks are identified, mutations are transferred to the humanized frameworks, and the resulting humanized mutant antibodies are retested for activity and developability. The most promising humanized mutant Abs are then conjugated with toxic payloads (see, e.g., paragraphs [0495-0496] above) to generate candidate anti-LRRC15 ADCs, which are then evaluated for stability, activity, and developability. Table 7 lists the better performing candidates, and additional details are presented in Example 23 (below).

[0566] Table 7. Selected anti-LRRC15 antibodies exhibit enhanced dissociation, enhanced internalization and increased cytotoxicity at acidic pH. VH = heavy chain variable domain; VL = light chain variable domain

[0567]

[0568] Tables 8-13 provide the detailed information of the constructs for specific purposes during the humanization process. And although the development candidate disclosed in Table 7 (above) is the most advanced, during the developability of the associated proteins disclosed herein, the applicant strives to improve, and many useful intermediate forms are produced. The developability modification includes those that tend to improve Ab solubility, reduce aggregation and optimize the modification of the in vivo half-life. Since the applicant has fully disclosed various tolerable and / or beneficial "developability modifications", those skilled in the art can routinely produce similar associated proteins with desired drug-like properties. The following table shows some of these developability constructs and the characteristics associated therewith.

[0569] Table 8. Selected humanized anti-LRRC15 binding proteins

[0570]

[0571] Table 9. Selected Histidine-substituted Anti-LRRC15 Chimeric RabMAbs

[0572]

[0573]

[0574] Table 10. Selected chimeric anti-LRRC15 RabMAbs

[0575] MYT Number Parent molecule HC Sequence ID LC sequence ID MYT5712 15G7 378 451 MYT5742 24D9 512 518 MYT8088 29F1 572 577

[0576] Table 11. Internalization of Abs in U87-MG at 10 nM or cytotoxicity at 20 nM at 24 hours (FOS = fold compared to samrotamab; cytotoxicity = % killing at 20 nM)

[0577]

[0578] Table 12. Affinity of selected binding proteins for LRRC15 positive cell lines SAOS2 and G292

[0579]

[0580]

[0581] Table 13. Binding of selected proteins to LRRC15 neg. cell line Ramos (measured by MFI)

[0582]

[0583]

[0584] Table 14. Shows selected antibodies (by MYT numbering) and component parts (by SEQ ID NO). VH = heavy chain variable domain; VL = light chain variable domain

[0585]

[0586]

[0587]

[0588]

[0589] 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 ABPCs specific for LRRC15 compared to control ABPCs specific for LRRC15.

[0590] As discussed herein, pH-engineered ABPCs specific for LRRC15 exhibit desirable properties of decreased LRRC15 binding at acidic pH (e.g., about pH 5.0 to about pH 6.4), but enhanced binding at higher, e.g., physiological pH (e.g., pH 7.4), which enhances the accumulation of the pH-engineered ABPCs in endolysosomes under physiological conditions.

[0591] pH-dependent binding to LRRC15 on cells

[0592] To demonstrate that pH-engineered ABPCs specific for LRRC15 bind to cell surface LRRC15 at neutral pH, a cell surface binding assay was performed. A panel of human cells that are LRRC15+ was assembled. Methods for identifying and quantifying gene expression (e.g., LRRC15) of a given cell line are known in the art and include, for example, consulting the Cancer Cell Line Encyclopedia (CCLE; portals.broadinstitute.org / ccle) to determine the expression level and / or mutation status of a given gene in a tumor cell line, rtPCR, microarray or RNA-Seq analysis, or staining of cells with antibodies known in the art (e.g., recombinant anti-LRRC15 antibody, Abcam catalog number ab150376 clone EPR8188 (2); Hi-Affi TMRecombinant rabbit anti-LRRC15 monoclonal antibody, Creative Biolabs catalog number MOR-2090 clone number DS2090AB, for LRRC15). Cells were seeded at approximately 5-10,000 per well in 150 μL of pH 7.4 medium and incubated at 37°C for 5 minutes at several doses (e.g., two-fold dilution series) ranging from 1 pM to 1 μM with one of the following antibodies: a known control ABPC specific for LRRC15 (e.g., antibody, samatuximab, hu139.10, huAD208.4.1, huAD208.12.1, 1-13C3, or 1-19G12); a pH-engineered ABPC specific for LRRC15; and an appropriate negative isotype control mAb (e.g., Biolegend purified human IgG1 isotype control recombinant antibody, catalog number 403501). Before the experiment began, the binding properties of all antibodies were verified using methods known in the art. After incubation for 5 minutes, the cells were fixed with 4% formaldehyde (20 minutes at room temperature) and incubated with appropriate fluorophore-labeled secondary antibodies (e.g., Thermo Fisher Scientific mouse anti-human IgG1 Fc secondary antibody, Alexa Fluor 488, catalog number A-10631) for 60 minutes. Unbound reagents were washed with a series of PBS washes, and the cell groups were imaged using confocal microscopy. When the images were analyzed, significant fluorescence could be observed on the surface of cells bound to known control ABPCs specific for LRRC15 and pH-engineered ABPCs specific for LRRC15, but little surface binding could be observed for isotype negative controls. In order to separate the effect of pH on surface binding, the same experiment was repeated twice, in which the primary antibody incubation was performed at sequentially decreasing pH (e.g., pH 6.5, pH 5.5, and pH 5.0). Analysis of the resulting confocal microscopy images can show significant fluorescence on the surface of cells that bind to all mAbs tested, except for the isotype negative control, and for pH-engineered ABPCs specific for LRRC15, this fluorescence decreases as pH decreases. Alternatively, the mean fluorescence intensity of the cells is analyzed by flow cytometry using methods known in the art. The dissociation constant KD of the analyzed antibodies on cells at neutral pH is determined by nonlinear regression methods known in the art (e.g., Scatchard plot). Overall, the results can show that the pH engineering process results in the generation of pH-engineered ABPCs that are specific for LRRC15 and are pH-dependent in their binding properties, and that the ABPCs bind more effectively at neutral pH than at more acidic pH.Other methods of assessing the pH dependence of pH-engineered ABPCs specific for LRRC15 are known in the art and include, for example, the use of flow cytometry to measure ABPC surface binding.

[0593] pH-dependent release of LRRC15 from cells

[0594] To demonstrate that pH-engineered ABPCs specific for LRRC15 are capable of releasing LRRC15 at low pH after binding at neutral pH, a variation of the cell surface binding assay described above was performed using methods known in the art (e.g., as generally described in Gera N. (2012) PLoS ONE 7(11):e48928). Briefly, appropriate LRRC15+ cell lines (passage number less than 25) were harvested and plated at 50,000 cells per well in U-bottom 96-well microplates. Three conditions were 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 pH-engineered ABPCs specific for LRRC15 and control ABPCs specific for LRRC15 were tested. According to the conditions tested, at pH 7.4 or 5.0, wash the cells twice with 200 μL of FACS buffer (1×PBS containing 3% fetal bovine serum). The purified protein samples were diluted into the FACS buffer of the appropriate pH and added to the cells, and allowed to bind on ice for one hour. After incubation with the primary antibody, the pH 7.4 and pH 5.0 conditions were washed twice as before, and then 100 μl of secondary rat anti-human Fc AF488 (Baijin Biotechnology Company 410706) or other appropriate antibodies (1:50 dilution) or anti-Myc-Tag mouse mAb-AF488 (Cell Signaling Technologies (Cell Signaling Technologies) 2279S) (1:50 dilution) was added to the FACS buffer of the appropriate pH and incubated on ice for 30 minutes. The pH 5.0 release conditions were washed twice with the FACS buffer of pH 7.4, then resuspended in the FACS buffer of pH 5.0 of 100 μ l, and incubated on ice for 30 minutes, then secondary staining was carried out in the FACS buffer of pH 7.4 as described for other conditions. As before, the plate was washed twice and resuspended in 1% paraformaldehyde in appropriate FACS buffer, so that they are fixed for flow cytometry analysis. All conditions were read on flow cytometer (Accuri C6, BD Biosciences). Combination was observed as FLI signal (as mean fluorescence intensity) relative to the transition of independent secondary staining.After data analysis, it can be determined that both the pH-engineered ABPCs specific for LRRC15 and the control ABPCs specific for LRRC15 effectively bind to the surface of LRRC15+ cells at neutral pH, but the pH-engineered ABPCs specific for LRRC15 bind poorly at pH 5.0; similarly, it can be determined that the pH-engineered ABPCs specific for LRRC15 bind at pH 7.4, but then release LRRC15 at pH 5.0.

[0595] Enhanced endolysosomal delivery of pH-engineered ABPCs specific for LRRC15 compared to control ABPCs specific for LRRC15 in LRRC15+ cells

[0596] To verify and demonstrate that ABPCs specific for LRRC15 achieve endolysosomal localization after cellular uptake, internalization assays were performed using methods known in the art (e.g., Mahmutefendic et al., Int. J. Biochem. Cell Bio., 2011). Briefly, a panel of human cells that highly express LRRC15 was assembled using methods known in the art as described herein. The cells were plated, washed three times with PBS, and incubated at 37°C for 60 minutes in a neutral pH medium supplemented with a known control ABPC specific for LRRC15 (e.g., as described herein), a pH-engineered ABPC specific for LRRC15, and an appropriate negative isotype control mAb (e.g., as described herein) at a concentration of 2 micrograms per milliliter. In a subset of cells, verification of antibody internalization and endosomal localization is performed using methods known in the art; for example, cells are fixed in 4% formaldehyde as described herein, permeabilized using TWEEN20 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, such as a fluorescent RAB11 antibody (RAB11 antibody, Alexa Fluor 488, 3H18L5, ABfinity TM Rabbit monoclonal antibody), stained with an appropriate fluorescently labeled anti-human secondary antibody (e.g., as described herein), and imaged using confocal fluorescence microscopy as described herein. Analysis of confocal images can be used to show that both pH-engineered ABPCs specific for LRRC15 and control ABPCs specific for LRRC15 are internalized and accumulated in endolysosomes.

[0597] To demonstrate that pH-engineered ABPCs specific for LRRC15 achieve enhanced endolysosomal accumulation relative to control ABPCs specific for LRRC15, a pHrodo-based internalization assay was performed using both known control ABPCs specific for LRRC15 (e.g., as described herein) and pH-engineered ABPCs specific for LRRC15. TM iFL (P36014, Thermo Fisher Scientific), a dye whose fluorescence increases as pH decreases, making its fluorescence level outside the cell at neutral pH lower than its fluorescence level inside the acidic pH environment of endolysosomes. Briefly, appropriate LRRC15+ cell lines (less than 25 passages) were suspended in their recommended culture medium (e.g., by cell bank or cell bank database ATCC, DSMZ or ExPASy Cellosaurus) and plated in 24-well plates at a density of 2,000,000 cells / mL and 1 mL per well. While keeping the cells on ice, 1 mL of 2x pHrodoiFL-labeled antibody (prepared according to the manufacturer's instructions) was added to each well, the wells were pipetted / mixed five times, and the plates were incubated on ice in a dark environment for 45 minutes. The same but separate plates were also incubated on ice, with ice being meant as a non-internalized negative control. After such incubation, the experimental plates were moved to a 37°C incubator, the negative control plates were kept on ice to slow or block internalization, and samples were taken at designated time points to generate an internalization time course. The samples were placed in a U-shaped bottom 96-well plate, and internalization was quenched by adding 200 μL / well of ice-cold FACS buffer. The plates were spun at 2000xg for 2 minutes, resuspended in 200 μL of ice-cold FACS buffer, spun again, and resuspended in FACS buffer for the second time. Finally, the samples were loaded into a flow cytometer, and the cell pHrodo fluorescence was read using an excitation wavelength and an emission wavelength (566nm and 590nm, respectively) consistent with the excitation and emission maxima of the pHrodo iFL red dye. After completing the flow cytometry experiments and analyzing the data, it was observed that cells treated with pH-engineered ABPCs specific for LRRC15 had a higher pHrodo iFL signal relative to known control ABPCs specific for LRRC15, indicating that the pH-engineered ABPCs specific for LRRC15 achieved enhanced endolysosomal accumulation relative to the control ABPCs specific for LRRC15.

[0598] Alternatively, to demonstrate that pH-engineered ABPCs specific for LRRC15 achieve enhanced endolysosomal accumulation relative to control ABPCs specific for LRRC15, a variation of the above experiment was performed. LRRC15+ cells were plated, washed three times with PBS and incubated for 60 minutes at 37°C in a neutral pH medium supplemented with pH-engineered ABPCs specific for LRRC15 or control ABPCs specific for LRRC15 at a concentration of 2 μg / mL. After incubation, cells were washed three times with PBS, fixed and permeabilized, and stained with a panel of appropriately selected antibodies that bind to late endosomal markers as well as lysosomes (e.g., RAB7 and LAMP1; Cell Signaling Technology, Endosomal Marker Antibody Sampling Kit No. 12666; Abcam, Anti-LAMP2 Antibody [GL2A7], ab13524). After primary antibody staining, cells were stained with an appropriate mixture of fluorescently labeled secondary antibodies (e.g., goat anti-human IgG (H&L) secondary antibodies (Alexa Fluor 647) catalog number A-21445 and Abcam goat anti-rabbit IgG H&L (Alexa Fluor 488) catalog number ab150077), imaged using confocal fluorescence microscopy, and the co-localization areas of signals from the LRRC15-specific antibody and endosomal markers were visualized and quantified. When the data were analyzed, it was revealed that the co-localization of endolysosomes and LRRC15-specific antibody signals in wells treated with pH-engineered ABPCs specific for LRRC15 was increased compared to wells treated with control ABPCs specific for LRRC15, and thus it was demonstrated that pH-engineered ABPCs specific for LRRC15 achieved enhanced endolysosomal accumulation relative to control ABPCs.

[0599] Increased LRRC15 antigen density in LRRC15+ cells after exposure to pH-engineered ABPCs specific for LRRC15 compared to control ABPCs specific for LRRC15

[0600] To demonstrate that treatment of cells with pH-engineered ABPCs specific for LRRC15 does not cause a detectable decrease in the level of LRRC15 on the surface of cells exposed to pH-engineered ABPCs specific for LRRC15, or that the treatment causes a lesser decrease in the level of LRRC15 on the surface of cells exposed to pH-engineered ABPCs specific for LRRC15 relative to control ABPCs specific for LRRC15, an antigen density study using flow cytometry was performed. Briefly, 4.0×10^5 cells expressing LRRC15 were plated per well in a 96-well plate in 100 μL of culture medium. Cells were treated with titers ranging from 1 pM to 1 μM of: i) pH-engineered ABPCs 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 at 37°C for 2 hours, at which point all cells are incubated at 4°C for 30 minutes with 200 nM of a second control ABPC (e.g., as described herein) that is fluorophore-labeled and specific for LRRC15 and that has a different epitope than the first control ABPC or pH-engineered ABPC specific for LRRC15 (as determined, for example, by competitive binding studies on the cells). After this 30-minute incubation, the mean fluorescence intensity (MFI) of all cells is read out using, for example, flow cytometry, using methods known to one of ordinary skill in the art. 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 can be generated using a commercially available quantitative kit (e.g., BD Biosciences PE Phycoerythrin Fluorescence Quantification Kit, catalog number 340495); the quantitative standard curve is created by following the manufacturer's instructions. Other methods of determining the absolute number of LRRC15 on the surface of cells are known in the art and include, for example, the use of radioactive isotope-labeled reagents. When the data is analyzed, it can be revealed that at at least one antibody concentration, cells treated with control ABPCs specific for LRRC15 experience a reduction in the level of LRRC15 on their surface, while cells treated with pH-engineered ABPCs specific for LRRC15 experience a significantly smaller reduction or no reduction at all relative to both the isotype control and the untreated control.

[0601] Example 3. Conjugation of pH-engineered ABPCs and control ABPCs with cytotoxic drugs

[0602] An antigen binding protein construct conjugate (ADC) was prepared, comprising the LRRC15 binding IgG described herein (hereinafter referred to as LRRC15-IgG) linked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc) linker (hereinafter referred to as LRRC15-IgG-DC). Conjugation of the antigen binding protein construct with vcMMAE began with partial reduction of LRRC15-IgG followed by reaction with maleimidocaproyl-Val-Cit-PABC-MMAE (vcMMAE). LRRC15-IgG (20 mg / mL) was partially reduced by addition of TCEP (2:1 molar equivalents of TCEP:mAb) followed by overnight incubation at 0°C. The reduction reaction was then warmed to 20°C. To conjugate all thiols, vcMMAE was added to a final vcMMAE:reduced Cys molar ratio of 1:15. The conjugation reaction was performed in the presence of 10% v / v DMSO and allowed to proceed at 20°C for 60 minutes.

[0603] After the conjugation reaction, an excess of free N (acetyl) -cysteine ​​(2 equivalents relative to the vcMMAE charge) was added to quench the unreacted vcMMAE to generate the Cys-Val-Cit-MMAE adduct. The Cys quenching reaction was allowed to proceed at 20°C for approximately 30 minutes. The Cys quenching reaction mixture was purified as follows. The above conjugation method can also be used to conjugate maleimidocaproyl monomethyl auristatin F (mcMMAF) to the antigen binding protein construct.

[0604] LRRC15-IgG-DC was purified using a batch purification method. The reaction mixture was treated with an appropriate amount of water-washed Bu-HIC resin (ToyoPearl; Tosoh Biosciences), i.e., seven weights of resin were added to the mixture. The resin / reaction mixture was stirred for an appropriate time, and the removal of the drug conjugate product was monitored by analytical hydrophobic interaction chromatography, and filtered through a crude polypropylene filter and washed with two bed volumes of buffer (0.28M sodium chloride, 7mM potassium phosphate, pH 7). The combined filtrate and rinse were combined and the product profile was analyzed by HIC HPLC. The combined filtrate and rinse were buffer exchanged into 15mM histidine (pH 6) by ultrafiltration / diafiltration (UF / DF) with 10 times the dialysis volume of 15nM histidine buffer.

[0605] A similar protocol 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 nanomoles) was diluted into 13.5 mL of reducing buffer containing 10 mM sodium borate pH 8.4, 2.5 mM EDTA, and a final antibody concentration of 1.11 mg / mL. 10mMTCEP solution (1.5 molar equivalents / antibody, 150 nanomoles, 15 microliters) was added, and the reduction mixture was heated in an incubator at +37°C for 1.5 hours. After cooling to room temperature, SG3249 was added as a DMSO solution (5 molar equivalents / antibody, 500 nanomoles in 1.5 mL DMSO). The solution was mixed at room temperature for 1.25 hours, then conjugated by adding N-acetylcysteine ​​(1 micromolar, 100 microliters, 10mM) and injected into the AKTA using a GE Healthcare HiLoadTM26 / 600 column filled with Superdex 200PG TMPure FPLC and eluted with 2.6 ml / min sterile filtered phosphate buffered saline (PBS). The fractions corresponding to the LRRC15-IgG-DC monomer peak were pooled, concentrated using a 15mL Amicon Ultracell 50KDaMWCO spin filter, analyzed and sterile filtered. Using a Phenomenex Aeris 3.6u XB-C18 150×2.1mm column on a Shimadzu Prominence system, eluted with a gradient of water and acetonitrile, UHPLC analysis of the LRRC15-IgG-DC reduced sample (SG3249 specific) at 280nm and 330nm showed a mixture of light and heavy chains attached to several SG3249 molecules, which is consistent with a drug / antibody ratio (DAR) of 1 to 4 SG3249 molecules per antibody. Using a Phenomenex Yarra 3u SEC-3000 300mm×4.60mm column on a Shimadzu Prominence system, eluted with a sterile filtered SEC buffer containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride, and 10% isopropanol (v / v), UHPLC analysis of the LRRC15-IgG-DC sample at 280nm showed a monomer purity of more than 90% with no impurities detected. UHPLC SEC analysis allowed the determination of a final LRRC15-IgG-DC yield of greater than 30%.

[0606] Alternatively, methods of conjugating toxins to antibodies via lysine residues are known in the art (e.g., see Catcott KC et al. (2016) Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates, MAbs 8:513-23). ​​In addition, methods similar to the above can be used to conjugate drugs and toxins to non-IgG forms, such as Vh-Fc, via disulfide bonds.

[0607] Example 4. Demonstration of enhanced cytotoxicity of a pH-engineered ABPC ADC specific for LRRC15 compared to a control ABPC ADC specific for LRRC15 in LRRC15+ cells

[0608] A panel of LRRC15+ cell lines expressing various antigen densities (e.g., as described herein) and LRRC15- cell lines selected using the methods described herein, and optionally, cells expressing transgenic LRRC15, such as HEK293 cells transfected with LRRC15 using methods known in the art (e.g., Expi293 TMExpression System Kit (Thermo Fisher Scientific Catalog No.: A14635), the cytotoxic activity of both pH-engineered ADCs specific for LRRC15 (e.g., pH-engineered LRRC15-IgG-DC) and control ABPC ADCs specific for LRRC15 (e.g., control ABPC LRRC15-IgG-DC) was evaluated individually. For validation purposes, all cell lines were tested for expression of LRRC15 prior to use using methods known in the art, such as qPCR, flow cytometry, mRNA RPKM, and antibody staining using anti-LRRC15 antibodies known in the art (e.g., described herein), followed by staining visualization using fluorescence microscopy, immunohistochemistry, flow cytometry, ELISA, or other methods known in the art. To evaluate the cytotoxicity of the compounds, cells were seeded at approximately 10-40,000 / well in 150 microliters of culture medium and then treated with graded doses of compounds from 1 pM to 1 μM at the beginning of the assay in quadruplicate. Cytotoxicity assays were performed 96 hours after the addition of the test compound. During the last 4 to 6 hours of incubation, fifty microliters of resazurin dye was added to each well to assess viable cells 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 extent of resazurin reduction of treated cells was compared to that of untreated control cells, and the percentage of cytotoxicity was determined. Alternatively, cytotoxicity was measured using a WST-8 kit according to the manufacturer's instructions (e.g., Dojindo Molecular Technologies catalog number CCK-8). The concentration IC50 at which half-maximal killing was observed was calculated using curve fitting methods known in the art. When the data were analyzed, it was determined that the pH-engineered ABPC ADC and the control ABPC ADC that were specific for LRRC15 had significant cytotoxicity to one or more LRRC15+ cell lines, but had lower toxicity to LRRC15- cells. It can also be determined that a pH-engineered ADC specific for LRRC15 is more cytotoxic to one or more LRRC15+ cell lines than a control ABPC ADC specific for LRRC15 because: a) it exhibits a greater depth of killing at one or more concentrations, or b) it exhibits a lower IC50, or c) it exhibits a higher ratio of its dissociation constant KD (as described herein) on cells at neutral pH divided by its IC50 on those same cells.

[0609] In addition, the cytotoxic activity of ABPCs specific for LRRC15 can be measured in a secondary ADC assay. Secondary ADC assays are known in the art (e.g., Moradec Catalog No. αHFc-NC-MMAF and Catalog No. αHFc-CL-MMAE, and instructions from the relevant manufacturers). Briefly, the assay is performed as in the previous paragraph, except that the ABPCs specific for LRRC15 are replaced with an ADC specific for LRRC15, and to assess the cytotoxicity of the compound, cells are seeded at approximately 10-40,000 per well in 150 microliters of culture medium and then treated at the start of the assay with graded doses of ABPCs specific for LRRC15 from 1 pM to 1 μM (final concentration in culture medium, premixed with Moradec Catalog No. αHFc-NC-MMAF secondary ADC reagent at a final concentration of 100 nM in culture medium and pre-incubated at 37° C. for 30 minutes before the mixture is added to the culture medium), in quadruplicate.

[0610] The cytotoxicity of the pH engineered ADC specific for LRRC15 and the control ABPC ADC specific for the LRRC15 conjugate, as well as the ABPC specific for LRRC15 in a secondary ADC assay were additionally measured by a cell proliferation assay using the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al., Cancer Research 62:5485-5488, 2002):

[0611] 1. Deposit a 100 μl aliquot of a cell culture containing approximately 104 cells (eg, LRRC15+ cells described herein) in culture medium into each well of a 96-well opaque-walled plate.

[0612] 2. Prepare control wells containing culture medium and no cells.

[0613] 3. ADC specific for LRRC15 was added to the experimental wells at a concentration range of 1 pM-1 uM and incubated for 1-5 days. Alternatively, in the secondary ADC assay, 100 nM secondary ADC reagent (final concentration in culture medium, Moradec catalog number αHFc-NC-MMAF) and ABPC specific for LRRC15 at a concentration range of 1 pM-1 uM (final concentration in culture medium) were premixed and pre-incubated at 37°C for 30 minutes, after which the mixture was added to the culture medium and incubated for 1-5 days.

[0614] 4. Equilibrate the plate to room temperature for approximately 30 minutes.

[0615] 5. Add a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well.

[0616] 6. Mix the contents on an orbital shaker for 2 minutes to induce cell lysis.

[0617] 7. Incubate the plate at room temperature for 10 minutes to stabilize the luminescent signal.

[0618] 8. Record and report luminescence in graphs as RLU = relative luminescence units.

[0619] Example 5. Demonstration of enhanced toxin release of pH-engineered ABPC ADCs specific for LRRC15 compared to control ABPC ADCs specific for LRRC15 in LRRC15+ cells

[0620] pH-engineered ADCs specific for LRRC15 (e.g., pH-engineered LRRC15-IgG-DCs) can also exhibit increased toxin release in LRRC15+ cells compared to control ABPC ADCs specific for LRRC15 (e.g., control ABPC LRRC15-IgG-DCs). After treating LRRC15+ cells with pH-engineered ABPC ADCs specific for LRRC15 and control ABPC ADCs as described herein, LC-MS / MS methods were used to quantify unconjugated (i.e., released) MMAE in 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 interval and a triple quadrupole mass spectrometer was used. For the detection of MMAE, an XBridge BEH Amide column (Waters, Milford, MA) was used with a mobile phase A of water (containing 5 mM ammonium formate and 0.1% formic acid) and a mobile phase B of 95:5 acetonitrile / water (0.1% formic acid and 1 mM ammonium formate) with a gradient of 0.25 mL / min at 40°C. The total duration of the chromatographic run was 12 minutes, with two MRM scans (718.5 / 686.5 and 718.5 / 152.1 amu) monitored. Deuterated (d8) MMAE (MCE MedChem Express, Monmouth Junction, NJ) was used as an internal standard. First, an equation for quantifying unconjugated MMAE in biological samples was derived by dividing the peak area of ​​each drug standard by the peak area obtained by the internal standard. The obtained peak area ratio is then plotted according to the concentration of the standard, and the data points are fitted to a curve using linear regression. Three QC samples are included in the low, medium and high ranges of the standard curve to evaluate the predictive power of the developed standard curve. The observed concentration of MMAE in the biological sample is then inferred using the obtained standard curve. In order to measure the MMAE concentration, the treated cell sample is precipitated and reconstructed to a final concentration of 250,000 cells / 100 μL in fresh culture medium. The sample is spiked with d8-MMAE (1 ng / mL), followed by cell lysis by adding 2 volumes of ice-cold methanol, follo...

Claims

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

0.

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

3. The ABPC of claim 2, wherein the ABD comprises: (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 wherein each HCVD and / or LCVD has one or more amino acids substituted with histidine, aspartic acid or glutamic acid; Optionally wherein the ABD is selected from 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:455) 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 OR Optionally, the ABD is selected from an antibody comprising a heavy chain polypeptide sequence and a light chain polypeptide sequence pair that are at least 95% identical to the sequence pairs shown in the following: SEQ ID NO: 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.

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

5. The ABPC of claim 1, wherein the first ABD comprises an HCVD comprising CDR1, CDR2, and CDR3 of one of the following: (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 has in common one or more amino acid positions substituted with histidine, aspartic acid, or glutamic acid; and / or wherein the first ABD comprises a LCVD, the LCVD comprising CDR1, CDR2 and CDR3 of one of the following: (a) SEQ ID NOs: 452-454; (b) SEQ ID NOs: 519-521; and (c) SEQ ID NOs: 578-580, optionally wherein each set of three CDRs has in common one or more amino acid positions substituted with histidine, aspartic acid or glutamic acid.

6. The ABPC according to any one of claims 1 to 5, wherein the first ABD comprises a HCVD of one of: (a) a HCVD comprising a sequence at least 90% identical to the sequence shown 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; (b) an HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises 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) HCVD comprising a sequence at least 90% identical to the sequence shown 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: a D position selected from 31, 56 and 99; and an E position selected from 59.

7. The ABPC of any one of claims 1 to 5, wherein the first ABD comprises an LCVD of one of: (a) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises 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 shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD comprises 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 at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

8. The ABPC of any one of claims 1 to 5, wherein the first ABD comprises a HCVD of one of: (a) an HCVD comprising a sequence at least 90% identical to the sequence shown 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 comprising a pair selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD includes histidine at two or more positions in SEQ ID NO:512 or SEQ ID NO:516; and (c) an HCVD comprising a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCVD includes histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO: 572 or SEQ ID NO: 576; and / or wherein the first ABD comprises an LCVD of one of the following: (a) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises histidine at two or more positions of SEQ ID NO:451 or SEQ ID NO:455 comprising a 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 LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD comprises histidine at two or more positions of SEQ ID NO:518 or SEQ ID NO:522; and (c) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:

578. A sequence that is at least 90% identical to the sequence shown in SEQ ID NO:581, wherein the LCVD includes histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO:577 or SEQ ID NO:

581.

9. The ABPC according to any one of claims 1 to 5, wherein the first ABD comprises one of: (a) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:378 or SEQ ID NO:382, wherein the HCVD includes 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 a LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD includes 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) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD includes 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 a LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD includes 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 shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCVD comprises aspartic acid (D) or glutamic acid (E) at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59; and / or an LCVD comprising a sequence at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

10. The ABPC of claim 1, wherein the first ABD comprises an HCVD comprising the 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 wherein the first ABD comprises a LCVD comprising the 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 wherein the first ABD comprises a HCVD and a LCVD, and the HCVD and the LCVD respectively comprise the HCDR and the LCDR present in the HCVD and the LCVD of one of 15G7, 24D9 and 29F1.

11. The ABPC of claim 1, wherein the first ABD comprises a HCVD comprising the sequence shown 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 the sequence shown 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 does not comprise (a) a HCVD comprising the sequence shown in SEQ ID NO: 378 or SEQ ID NO: 382 and a LCVD not comprising the sequence shown 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 SEQ ID NO: or (b) an LCVD of SEQ ID NO:451 or SEQ ID NO:455 and an HCVD that does not comprise the sequence shown 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. The ABPC according to any one of claims 1 to 11, wherein after the ABPC is internalized by the target mammalian cell, the ABPC is degraded in the target mammalian cell; optionally wherein the ABPC further comprises a conjugated toxin, radioisotope, drug or small molecule.

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

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

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

16. The ABPCs according to any one of claims 1 to 15, wherein the ABPCs cause less or no detectable decrease in the level of LRRC15 presented on the surface of the target mammalian cells compared to the same amount of control ABPCs.

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

18. A composition comprising an effective amount of an antigen binding protein construct (ABPC), the ABPC comprising: a first ABD capable of specifically binding to LRRC15 or an epitope of LRRC15 presented on the surface of a target mammalian cell; and a conjugated toxin, radioisotope, drug or small molecule, optionally wherein: (a) the first ABD has a faster off-rate at a pH of about 4.0-6.5 than at a pH of about 7.0-8.0; or the first ABD has a dissociation constant (KD) at a pH of about 4.0-6.5 that is greater than the KD at a pH of about 7.0-8.0; and (b) the composition provides one or more of the following: (i) increased toxin release in the target mammalian cell compared to a composition comprising the same amount of a control ABPC; (ii) increased target mammalian cell killing compared to a composition comprising the same amount of a control ABPC; and (iii) increased endolysosomal delivery in the target mammalian cell compared to a composition comprising the same amount of a control ABPC.

19. A composition according to claim 18, wherein the first ABD comprises a heavy chain variable domain (HCVD) of one of the following: (a) 15G7; (b) 24D9; and (c) HCVD of 29F1, each HCVD optionally having one or more amino acids substituted by histidine, and optionally wherein the 29F1 HCVD has one or more amino acids substituted by aspartic acid or glutamic acid; and / or wherein the first ABD comprises a light chain variable domain (LCVD) of one of the following: (a) 15G7; (b) 24D9; and (c) LCVD of 29F1, each LCVD optionally having one or more amino acids substituted by histidine, optionally wherein the 29F1 LCVD has one or more amino acids substituted by aspartic acid or glutamic acid.

20. The composition of claim 19, wherein the ABD comprises (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 wherein each HCVD and / or LCVD has one or more amino acids substituted with histidine, aspartic acid or glutamic acid; Optionally wherein the ABD is selected from 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:455) 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 OR Optionally, the ABD is selected from an antibody comprising a heavy chain polypeptide sequence and a light chain polypeptide sequence pair that are at least 95% identical to the sequence pairs shown in the following: SEQ ID NO: 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 composition of claim 18, wherein the HCVD comprises one of: (a) HCVD of 15G7, comprising the sequence shown in SEQ ID NO: 378 or SEQ ID NO: 382; (b) HCVD of 24D9 comprising the sequence shown in SEQ ID NO:512 or SEQ ID NO:516; and (c) HCVD of 29F1 comprising the sequence shown in SEQ ID NO: 572 or SEQ ID NO: 576; and / or The LCVD comprises one of the following: (a) LCVD of 15G7, comprising the sequence shown in SEQ ID NO:451 or SEQ ID NO:455; (b) an LCVD of 24D9 comprising the sequence shown in SEQ ID NO:518 or SEQ ID NO:522; and (c) an LCVD of 29F1 comprising the sequence shown in SEQ ID NO: 577 or SEQ ID NO: 581; Optionally wherein each HCVD and / or LCVD has one or more amino acid positions substituted with histidine, aspartic acid or glutamic acid.

22. The composition of claim 18, wherein the first ABD comprises an HCVD comprising CDR1, CDR2 and CDR3 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 has in common one or more amino acid positions substituted with histidine, aspartic acid or glutamic acid; and / or wherein the first ABD comprises a LCVD, the LCVD comprising CDR1, CDR2 and CDR3 of one of the following: (a) SEQ ID NOs: 452-454; (b) SEQ ID NOs: 519-521; and (c) SEQ ID NOs: 578-580, optionally wherein each set of three CDRs has in common one or more amino acid positions substituted with histidine, aspartic acid or glutamic acid.

23. The composition of any one of claims 18 to 22, wherein the first ABD comprises an HCVD of one of: (a) a HCVD comprising a sequence at least 90% identical to the sequence shown 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; (b) an HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD comprises 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) HCVD comprising a sequence at least 90% identical to the sequence shown 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: a D position selected from 31, 56 and 99; and an E position selected from 59.

24. The composition of any one of claims 18 to 22, wherein the first ABD comprises a LCVD of one of: (a) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises 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 shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD comprises 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 at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

25. The composition of any one of claims 18 to 22, wherein the first ABD comprises an HCVD of one of: (a) an HCVD comprising a sequence at least 90% identical to the sequence shown 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 comprising a pair selected from the group consisting of: 34,53; 34,104; 34,105; 34,106; 53,104; 53,105 and 53,106; (b) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD includes histidine at two or more positions in SEQ ID NO:512 or SEQ ID NO:516; and (c) an HCVD comprising a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 572 or SEQ ID NO: 576, wherein the HCVD includes histidine, aspartic acid, or glutamic acid at two or more positions in SEQ ID NO: 572 or SEQ ID NO: 576; and / or wherein the first ABD comprises an LCVD of one of: (a) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD comprises histidine at two or more positions of a pair of positions in SEQ ID NO:451 or SEQ ID NO:455 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) a LCVD comprising a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD includes histidine at two or more positions in SEQ ID NO:518 or SEQ ID NO:522; and (c) an LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:577 or SEQ ID NO:581, wherein the LCVD includes histidine, aspartic acid or glutamic acid at two or more positions in SEQ ID NO:577 or SEQ ID NO:

581.

26. The composition of any one of claims 18 to 22, wherein the first ABD comprises one of: (a) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:378 or SEQ ID NO:382, wherein the HCVD includes 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 a LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:451 or SEQ ID NO:455, wherein the LCVD includes 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) a HCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:512 or SEQ ID NO:516, wherein the HCVD includes 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 a LCVD comprising a sequence at least 90% identical to the sequence shown in SEQ ID NO:518 or SEQ ID NO:522, wherein the LCVD includes 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 shown in SEQ ID NO:572 or SEQ ID NO:576, wherein the HCVD comprises aspartic acid (D) or glutamic acid (E) at one or more positions in SEQ ID NO:572 or SEQ ID NO:576 selected from the group consisting of: a D position selected from 31, 56 and 99; and an E position selected from 59; and / or an LCVD comprising a sequence at least 90% identical to the sequence shown 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: a D position selected from 27, 28, 31, 52 and 56; and an E position selected from 51 and 56.

27. The composition of claim 18, wherein 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, 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 wherein the first ABD comprises a LCVD comprising the 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 wherein the first ABD comprises a HCVD and a LCVD, and the HCVD and the LCVD respectively comprise the HCDR and the LCDR present in the HCVD and the LCVD of one of 15G7, 24D9 and 29F1.

28. The composition of claim 18, wherein the first ABD comprises an HCVD comprising the 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 the 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 does not comprise (a) a HCVD comprising the sequence shown in SEQ ID NO:378 or SEQ ID NO:382 and a LCVD that does not comprise the sequence shown 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) a LCVD of SEQ ID NO:451 or SEQ ID NO:455 and a HCVD that does not comprise the sequence shown 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 NOs:441-442, SEQ ID NOs:443-444, SEQ ID NOs:445-446, SEQ ID NOs:447-447, SEQ ID NOs:448-449, SEQ ID NOs:459-460, SEQ ID NOs:461-462, 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. NO:444 or SEQ ID NO:

450.

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

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 wherein the increased toxin release is at least 20%, 50%, 2-fold or 5-fold.

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

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

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

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

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

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

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

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

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

40. The ABPC according to claim 39, wherein the ABPC is selected from the group consisting of antibodies, VHH-scAb, VHH-Fab, bi-scFab, F(ab')2, bifunctional antibodies, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common light chain of knobs and holes, knobs and holes assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, kappa lambda 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, Triabody, Minibody, Microbody, TriBi Microbody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scdiabody-Fc, Diabody-Fc, Tandem scFv-Fc, VHH-Fc, Tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, Dock-Lock Antibody, ImmTAC, IgG-IgG Conjugate, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, Fibronectin, DEP Conjugate, PROTAC, and PROTAB.

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

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

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

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

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

46. A method of inducing cell death in cancer cells and / or CAFs in a subject, wherein the cancer cells and / or CAFs have a predetermined level of LRRC15 or an epitope of LRRC15 presented on their surface, the method comprising administering a therapeutically effective amount of the ABPCs or composition according to any one of claims 1 to 43 to a subject identified as having a cancer characterized by a population of cancer cells and / or CAFs, thereby inducing cell death in the cancer cells and / or CAFs.

47. A method of reducing the risk of developing metastasis 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 presenting a predetermined level of LRRC15 or an epitope of LRRC15 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 a population of said cancer cells, CAFs and / or stromal cells, thereby reducing the risk.

48. An anti-LRRC15 antibody comprising: (a) a heavy chain variable domain (HCVD) and a light chain variable domain (LCVD), the HCVD and the LCVD comprising a sequence that is 90% identical to the HCVD sequence and the LCVD sequence shown in one of the following sequence pairs, and comprising heavy chain CDRs and light chain CDRs that are identical to the HCVD sequence and the LCVD sequence shown 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: SEQ ID NOs: 84 and 177 and SEQ ID NOs: 576 and 625; or (b) HCVD and LCVD, the HCVD and the LCVD comprising a sequence that is 95% identical to the HCVD sequence and the LCVD sequence shown in one of the following sequence pairs, and comprising heavy chain CDRs and light chain CDRs that are identical to the HCVD sequence and the LCVD sequence shown 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: NO:576 and 625; or and (c) 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, SEQ ID NOs: 576 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: 731 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, optionally wherein position 205 of the light chain sequence of the antibody comprises an amino acid other than cysteine.

49. An anti-LRRC15 antibody according to claim 48, comprising a heavy chain sequence and a light chain sequence having a sequence as shown 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, optionally wherein position 205 of the light chain sequence of the antibody comprises an amino acid other than cysteine.

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

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