Identification of HLA epitopes targeted by the immune system

Abstract

Provided herein are antibodies or modified antibodies that, alone or in combination, bind a human leukocyte antigen (HLA; e.g., A*01 or A*01:01) and block binding to at least 50% of amino acids within a face of the HLA or block binding of at least 50% (including at least 75%, at least 85%, at least 90%) of DSAs (e.g., in a biological sample of a subject). Also provided are nucleic acids encoding the one or more antibodies or modified antibodies, compositions and kits comprising the one or more antibodies or modified antibodies, and methods of using the one or more antibodies to reduce an immune response in a transplant recipient, treat a donor organ or tissue, or screen a transplant recipient.

Description

[0001] IDENTIFICATION OF HLA EPITOPES TARGETED BY THE IMMUNE SYSTEM

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application No. 63 / 625,797 filed January 26, 2024, which is incorporated by reference herein in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. AI142737 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING STATEMENT

[0007] The instant application contains a Sequence Listing in XML format. The Sequence Listing, named 035979-1476610-221WOl.xml, which was created on January 23, 2025, is 337 Kilobytes in size, and is hereby incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] In the context of organ transplants, antibodies made by a recipient directed against the donor’s Human Leukocyte Antigen (HLA) proteins contribute to antibody-mediated rejection. These donor-specific antibodies (DSA) are a major barrier to the success of organ transplantation. More than half of the patients on the US renal transplant waitlist, for example, are sensitized to HLA, particularly multiparous women and patients sensitized to prior transplants. Chronic antibody-mediated rejection eventually results in transplant failure. HLA proteins are highly polymorphic and comprise amino acid epitopes with varying immunodominance. Thus, it is difficult to evaluate the likelihood of antibody-mediated rejection (AMR) prior to transplant to effectively allocate available organs and to treat or prevent antibody-mediated rejection post-transplant.

[0010] SUMMARY

[0011] Identified herein are epitopes of donor HLA molecules recognized by donor-reactive B cells and antibodies and modified antibodies with paratopes that block binding of donorspecific antibodies (DSA), which are produced by the transplant recipient, that target the mismatched donor HLA molecules. The detailed analysis provided useful antibodies or modified antibodies that, alone or in combination, bind a human leukocyte antigen A* 01 or A*01 :01 and block binding to at least 50% (including at least 75%, at least 85%, at least 90%) of amino acids within a face of the HLA A*01 or A*01 :01 or block binding of at least 50% (including at least 75%, at least 85%, at least 90%) of DSAs (e.g., in a biological sample of a subject).

[0012] The antibodies and modified antibodies provided herein can be full-length or portions thereof. For example, the antibodies and modified antibodies can be chimeric antibodies or portions thereof, single chain antibodies or portions thereof, single chain variable fragments (scFvs), Fab' fragments, F(ab')2 fragments, or bispecific antibodies or portions thereof (e.g., that bind to and / or bind block multiple binding sites within the face of the HLA). The modified antibodies can include one or more modifications in the Fc region of the antibody.

[0013] Also provided are nucleic acids encoding the antibodies or modified antibodies as described herein and compositions and kits containing one or more antibodies or modified antibodies. The kits optionally comprise one or more antibodies or modified antibodies and / or one or more human leukocyte antigen molecules or portion thereof, either of which can be attached to a solid support (e.g., a mobile solid support such as beads). Also provided are pharmaceutical compositions and kits comprising the pharmaceutical compositions and, optionally, a therapeutic device for delivery to a subject.

[0014] When the antibodies or modified antibodies bind amino acids within the face of the HLA, they block or prevent the antibody binding site and reduce or prevent AMR. Thus, provided herein are various compositions of matter and methods for screening and methods for treatment. More specifically, provided is a method of reducing an immune response in a transplant recipient to a donor organ comprising administering to the transplant recipient the one or more antibodies or modified antibodies identified herein in an amount effective to reduce the immune response (e.g., AMR) in the transplant recipient as compared to a control immune response in the absence of administration of the one or more antibodies or modified antibodies. Treatment can be performed before, concurrent with, or after transplantation or can be performed when clinical signs of AMR are detected. The antibodies or modified antibodies can be administered to the subject along with immunosuppressants (i.e., before, after, or concurrently with one or more immunosuppressants) or instead of one or more immunosuppressants .

[0015] Also provided is a method of screening a biological sample from a subject to identify one or more antibodies or modified antibodies that together or alone block binding of at least 50% (at least 75%, at least 85%) of donor specific antibodies to human leukocyte antigen A*01 or A*01 :01 in the subject. Such a screening method can be performed before treatment so as to personalize the selection of the antibodies or modified antibodies that could be used to treat the particular subject. A method of screening a transplant recipient before or after transplantation can be used to characterize the donor specific antibodies produced by the subject and this information can be used to define specific HLA epitopes recognized by the transplant recipient’s immune system. The screening method comprising obtaining a blood sample (e.g., whole blood, plasma, or serum) from the transplant recipient; contacting the blood sample the one or more antibodies or modified antibodies; and determining whether the one or more antibodies or modified antibodies block binding of at least 50% (e.g., at least 75%, at least 85%) of the subject’s donor specific antibodies to the human leukocyte antigen A*01 or A*01:01. The methods can further include contacting the antibodies or modified antibodies with human leukocyte antigens A*01 or A*01 :01 prior to the contacting of the blood sample followed by determining whether the one or more antibodies block binding of the subject’s donor specific antibodies to the human leukocyte antigen A*01 or A*01:01.

[0016] A method of treating a donor organ or tissue prior to transplantation is also provided to reduce binding of donor specific antibodies in a transplant recipient comprising contacting the donor organ with the one or more antibodies or modified antibodies.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a schematic showing the production and selection of recombinant monoclonal antibodies (rmAbs). AlloHLA-specific B cells are sorted as single cells into hyptonic lysis buffer in 384 well plates. B cell cDNA is generated by adding high-capacity RT reaction mix. cDNA is used as a template for two 1 st round PCR reactions using primers designed to amplify either IgHV genes rearranged as IgM, IgG, or IgA isotypes, or light chains (Kappa and Lambda PCRs are multiplexed). An aliquot of the 1st round PCR product is used as a template for a second, semi-nested PCR reaction. The resulting amplicons are mixed with linearized expression vectors designed to promote the expression of heavy chain variable genes as hlgGl and either Kappa or Lambda chains. The sequence dependent, directional ligation independent cloning strategy allows light chain multiplexing, eliminating! Kappa and lambda cross ligation. Chemically competent Top 10 E. coli are transformed with the cloning mixture and grown overnight in 8 deep well 96 blocks. Plasmids are isolated by alkaline lysis in a 96 well format then re-arrayed into 384 well plates while preserving the originating well position. IgH and IgL chains are co-transfected into 1ml pilot cultures of 293 Free Style cells. After 3 days, the conditioned media is harvested, cleared by centrifugation, and assayed for the presence of Ab using an HLA cytometric bead array. Indexed arraysequencing of IgHV and IgLV is performed. After selecting rmAbs for functional analyses, plasmids encoding selected rmAbs are isolated as single plasmids and then Sanger sequenced to confirm IgHV and IgLV usage and to assign rmAbs to clonal lineages. Hie rmAbs are moved into large scale Ab production for downstream functional assays (e.g. SPR). Plasmids can easily be modified to eliminate the IgGl Fc to generate Fab fragments, or subcloned into alternative vectors to produce chimeric antibodies or modified antibodies. Time requirements indicated.

[0019] FIGs. 2A-2D shows CryoEM data collection and reconstruction for the L02 / M07 / HLA-A*01 :01 complex. FIG. 2A is a representative cryoEM image of L02 / M07 / HLA-A*01 :01 complex. FIG. 2B shows fourier shell correlation (FSC) curves of half map FSCs calculated in cryoSPARC and model-map FSC calculated in Phenix. The 0.143 FSC cutoff is indicated by a dashed line. FIG. 2C shows reconstruction colored by local resolution. FIG. 2D shows representative fit of segments of the atomic model to the reconstructed density map.

[0020] FIGs. 3A-3M show that intragraft and circulating B cells exhibit features of antigen- driven selection against donor HLA-A*01:01. FIGs. 3A, 3B and 3C are images of immunofluorescent histology of a rejected kidney (KD) allograft from recipient N006 showing CD4+ T and CD 19+ B cells (FIG. 3 A), proliferating Ki67+ CD 19+ B cells (FIG. 3B), and CD19+, CD38+, intracellular IgH+ ASCs (FIG. 3C). DAPI nuclear staining is also shown in FIG. 3A, FIG. 3B and FIG. 3C. FIGs. 3D and 3E are graphs showing flow cytometry analysis of recipient N006 kidney allograft to identify dump negative (CD3 / CD14 / CD56neg) B lineage (CD 19+) subsets including antibody secreting cells (ASCs) (CD27+CD38+), naive (NAV, CD27negIgD+), Bmem (MEM, CD27+IgDneg) and CD27negIgDneg DN B cells (FIG. 3D) and CD71 (FIG. 3E) to identify activated ASCs (CD71+CD27+CD38+) and activated B cells (CD71+dump negativeCD19+). FIG. 3F shows alluvial plots showing lineage relatedness between ASCs, DN B cells, and Bmem cells isolated from recipient N006 KD allograft. Shared lineages (ribbons) ranked by size in each B cell subset with lineages shared between all 3 subsets or between any two subsets. Numbers of sorted cells and non-singleton lineages for each subset provided. %D50 defined as: (# lineages in top 50% of subset A / # of lineages in the top 50% of subset B) x 100. See Methods in examples for details. FIG. 3G shows a circos plot showing connections between lineages of B cell subsets isolated from recipient N006 KD allograft and peripheral blood (PBL). Size-ranked lineages representing the top 20% of size-ranked sequences within a given subset are colorized. See Methods in examples for details. FIG. 3H shows a scatterplot comparing size of B cell lineages in recipient N006 KD (x-axis) and PBL (y-axis). Lineage size shown as % sequences of that lineage within KD or PBL. Lineages not detected in a tissue reported as 0%. FIG. 31 is a graph showing trajectory of anti-HLA-A*01:01 DSA response in recipient N006 based upon clinical HLA-Ab testing. Clinical index diagnosis of AMR and timing of transplant nephrectomy indicated. FIG. 3 J show fluorochrome-labeled recombinant HLA-A*01:01 tetramer binding to CD19+IgDneg B cells isolated from recipient N006 kidney and blood. Dual tetramer-stained cells (gated population) were index-sorted for single cell rmAb production. FIG. 3K is a graph showing binding to HLA-A*01 :01 -coated microbeads by 50 rmAbs from single-cell sorted HLA-A*01:01-specific B cells (see FIG. 3 J) or human rmAbs specific for influenza HA protein (negative control, (NC)). Data reported as geometric mean fluorescent intensity (gMFI). FIG. 3L and 3M are graphs showing mutation frequencies (FIG. 3L) and isotype distribution (FIG. 3M) in KD and PBL B cell derived IgVH sequences that were assigned to A*01 :01-specific B cell lineages. Violin plots (FIG. 3L) showing IgVH-region nucleotide (NT) mutation frequency distributions with median number of mutations in each bulk subset and rmAb subset shown.

[0021] FIG. 4 shows an experimental overview and IgVH-Seq analyses of kidney and blood B cells and ASCs. FIG. 4A is a schematic showing sample acquisition and experimental design. B cells and ASCs, isolated from a rejected transplanted kidney and peripheral blood of kidney transplant recipient N006, were sort-purified for bulk BCR IgVH sequencing and generation of single A*01 :01-specific rmAbs. rmAbs were sequenced (IgVH and IgVL), tested for specificity, reactivity and affinity, and used in structural studies to identify binding sites (epitopes) on HLA-A*01:01. LC-MS / MS was used to analyze A* 01 :01 -binding affinity-purified polyclonal Abs (appAbs) from recipient N006 plasma. FIG. 4B shows flow cytometric analysis of B cells from rejected kidney showing gating and sort strategy used for IgVH-Seq repertoire analysis of bulk DumpnegCD19+ B lineage cells that were subdivided into ASCs (CD27+CD38+) and non- ASCs. Non-ASCs were further divided into naive (NAV, IgD+CD27neg), Bmem (MEM, CD27+IgDneg), and IgDnegCD27neg DN B cells. FIG. 4C shows alluvial plots depicting connections between lineages of Bmem isolated from the lung and blood (PBL, left panel) or small intestine (SI) and PBL (middle panel) from an organ donor, or the rejected kidney and PBL of transplant recipient N006 (right panel). Lineages defined as having an identical IGHV and IGHJ gene segment annotation, identical HCDR3 length, and HCDR3 nucleotide sequence identity of >85%. Shared lineages, represented as ribbons with individual lineages ranked by size in each subset. Numbers of sorted cells for IgVH sequencing, numbers of unique non-singleton lineages and %D50 are indicated. %D50 = (# lineages in top 50% of subset A / # of lineages in the top 50% of subset B) x 100. FIG. 4D shows isotype distribution as determined by IgVH-Seq for KD and PBL bulk CD19+IgDneg B cell and ASC subsets as defined in panel B. FIG. 4E shows comparison of IgVH mutation frequencies in bulk-sorted CD19+IgDneg KD and PBL B cells and ASCs as defined in panel B.

[0022] FIGs. 5A-5D show the characterization of A*01 :01 -specific B cells and ASCs. FIGs. 5A and 5B show flow cytometric analysis of B cells from rejected KD (recipient N006), showing gating to identify A*01:01-specific B cells (FI. 5A) and phenotype (FI. 5B) of single index-sorted A*01:01-specific kidney and blood B cells used to clone and express 50 A*01:01-specific rmAbs. FIG. 5C is a dot plot depicting number of sequences and number of V-region nucleotide (NT) mutations for the 8 largest A* 01 :01 -specific lineages in the IgVH-Seq repertoire (n=3851 sequences), divided by tissue and cell subset. Subsets defined as in Figure 4B. FIG. 5D shows violin plots showing IgVH-region NT mutation distributions for the 8 largest A*01:01-specific lineages (n=3851 sequences) with median number of NT mutations per lineage indicated.

[0023] FIGs. 6A-6J shows alloreactive B cells undergo clonal expansion and affinity maturation and produce antibodies of high affinity for HLA-A*01:01. FIGs. 6A-6C show phylogenetic trees showing 3 representative IgVH clonal lineages of A*01:01-specific B cells and ASCs. Tree edges trace IgVH mutations from the inferred UCA (black node) through inferred mutation nodes (light grey nodes) and observed nodes (grey-scale symbols). Observed node shape denotes B cell subset(s) and tissue of origin assigned to each IgVH node (see panel callout). Nodes containing IgVH sequences from A*01:01-specific rmAbs indicated with callout of the rmAb clone name. IgVH-region NT mutation accumulation in phylogenetic trees is indicated to the right of the trees. FIG. 6D shows analysis of the A*01 :01-specific IgVH-Seq repertoire (n=4142 sequences) from the bulk kidney and blood B cell subset IgVH database described in Fig. 3F-H, 3L-M. Percentages of sequences assigned to the 14 different A*01:01-specific expanded lineages provided with callouts for individual clonal lineages representing ≥2.5% of the A*01:01-specific IgVH sequence database. FIG. 6E shows distribution of different B cell subsets within the 14 A*01:01 -specific B cell clonal lineages. FIG. 6F shows binding kinetics of A*01:01-specific rmAbs E07 and L02 to recombinant HLA-A*01:01. Binding reported as a function of increasing rmAb concentration, fit as 1 : 1 Langmuir curves by localized surface plasmon resonance (L-SPR). Affinity of binding reported as equilibrium constant (KD). FIG. 6G shows affinities (KD) of 50 A*01:01-specific rmAbs binding to A*01:01. Dotted line indicates limit of detection (KD 4.3 p.M). FIG. 6H shows binding to A*01:01 by representative Lin4 (imAb D01) and Lin12 (rmAb L02) A*01:01-specific rmAbs and synthetic rmAbs produced using the sequences of the inferred Lin4 and Lin12 UCAs. Data shown as increasing concentrations of the rmAbs (x- axis) and gMFI of binding to A*01 :01 cytometric beads (y-axis). L-SPR derived KD values provided. FIG. 61 shows paired analysis (n=36 pairs) showing L-SPR calculated KD for A*01 :01 by cloned A*01 :01-specific rmAbs and the synthetic UCA rmAb that was predicted to be the non-mutated ancestor of each of the cloned somatically mutated rmAbs. FIG. 6J shows paired analysis (n=9 pairs representing distinct A*01:01-specific B cell lineages) showing L-SPR calculated KD for A*01 :01 by cloned A*01 :01-specific rmAbs (showing rmAb with the highest affinity in each lineage) and the synthetic UCA rmAbs for each lineage. Statistical analyses in (FIGs. 6I-6J) performed using the Wilcoxon matched-pairs signed rank test.

[0024] FIGs. 7A-7F shows a direct linkage between donor specific antibodies found in circulation and ASCs and memory B cells isolated from kidney and blood. FIG. 7A shows phylogenetic tree showing Lin4 A*01 :01-specific B cells and ASCs from kidney and blood. See description of tree symbols and lines in Figure 6A. FIGs. 7B-7E show alignment of plasma proteomics data to A*01 :01 -specific rmAb sequences. Peptide sequences derived from LC-MS / MS analysis of enzymatically digested A*01:01-specific polyclonal appAbs from recipient N006 were aligned against sequences of Lin4 (FIGs. 7B-7C), Linl4 (FIG. 7D), and Lin6 (FIG. 7E) A*01:01-specific rmAbs cloned from B cells isolated from recipient N006. Exact peptide matches are plotted by position within the rmAb VDJ AA sequence. Peptide sequences overlapping the rmAb HCDR3 shown. L-SPR-calculated KD of the A*01 :01-specific rmAbs indicated. FIG. 7F is a column plot showing number of IgVH sequences derived from bulk sorted blood and kidney ASCs in 11 A*01:01-specific clonal lineages. A*01:01-specific lineages containing AA sequences that were also detected in the A*01 :01-specific polyclonal appAb proteome include Lin4, Linl4, Lin3 and Lin6.

[0025] FIGs. 8A-8C show lineage-specific patterns of HLA-A reactivity by rmAbs. FIG. 8A shows an amino acid (AA) sequence alignment of HLA-A self-alleles (A*24:02 and A*30:01) expressed by recipient N006 and the donor (A*01:01) kidney. Shown are AA residues mismatched between donor and recipient HLA-A alleles. Mismatch type (1MM or 2MM) and solvent accessible surface area (<50 A2 or ≥50 A2 ) are indicated for each mismatched residue. The AH-PBG region, which is defined as a-chain residues 50-85 and 138-175, is indicated with black helices above the sequence. FIG. 8B shows binding of 50 A*01 :01 -specific rmAbs (grey symbols) and 16 negative control (NC) rmAbs (specific for influenza hemagglutinin IgG186, dark grey symbols concentrated at the lowest gMFI level for each A*01:01 bead) to microbeads arrayed with 21 individual HLA-A proteins. Data reported as gMFI. FIG. 8C is a heatmap showing the most common HLA-A reactivity patterns for 50 A*01:01-specific rmAbs. HLA reactivity patterns for individual rmAbs were determined by calculating the net gMFI (subtracting the mean NC gMFI from the allelespecific gMFI of each A*01:01 specific rmAb) and then converting the net gMFI into positive (net gMFI ≥40,000, black squares) or negative (net gMFI <40,000, white squares) binary variables.

[0026] FIGs. 9A-9I shows immunodominant reactivity patterns within the alloreactive B cell repertoire and demonstrate that rmAbs E07, L02 and M07 recognize distinct structural epitopes in A*01:01. FIG. 9A shows HLA-A reactivity profile by 50 A*01:01-specific rmAbs cloned from B cells isolated from recipient N006 KD and blood. Data reported as net gMFI of rmAb binding to microbeads displaying 21 individual HLA-A alleles following normalization against a panel of negative (influenza-specific) control human rmAbs (see Methods). Data clustered using Euclidean distance. FIG. 9B shows comparison between HLA reactivity profiles of 50 A* 01 :01 -specific rmAbs and circulating affinity-purified A* 01 flspecific appAb isolated from recipient N006 plasma. Upper plot shows number of rmAbs that bound (net gMFI ≥40,000) to each HLA-A protein bead. Bottom plot shows net gMFI of appAb (diluted 2-fold from 5 pg / mL to 2 ng / mL) binding to each HLA-A protein bead. FIG. 9C is a pie chart depicting the most common HLA reactivity patterns for 50 A*01:01-specific rmAbs cloned from recipient N006 kidney and blood B cells. rmAb HLA reactivity patterns were clustered by converting net gMFI of each allele into binary variables (net gMFI ≥40,000 = positive or net gMFI <40,000 = negative). FIG. 9D is a pie chart depicting the most common HLA reactivity patterns for the 14 A*01:01-specific IgVH lineages (n=4142 sequences). For each clonal lineage, a dominant pattern of anti-HLA reactivity was assigned by identifying the specific HLA-A proteins bound the rmAbs associated with each clonal lineage. FIGs. 9E-9G show structures showing IgH and IgL chains of kidney Bmem cell derived A*01:01-specific rmAbs E07 (FIG. 9E), L02 (FIG. 9F) and M07 (FL 9G) bound to HLA-A*01:01. HLA-A*01 :01 a-chain, 02-microglobulin (02m) and peptide are indicated in each structure. Number of A* 01 :01 a-chain AA residues contained within each epitope and the amount of A*01 :01 a-chain surface area buried upon binding of each rmAb indicated. FIG. 9H shows merged, simulated structure showing binding of rmAbs E07, L02, and M07 to HLA-A*01 :01. FIG. 91 shows merged, simulated structure showing binding of rmAbs E07 and L02 to HLA-A*01 :01. Statistical analysis in FIG. 9C tested a null hypothesis that there would be 7 different patterns of reactivity corresponding to the 7 solvent-accessible and double-mismatched residues expressed by donor A*01:01 relative to recipient HLA-A*24:02 and A*30:01. Data analyzed using a binomial test with an expected frequency for an A*01 :01-monospecific pattern of 14.3%. The confidence interval of the proportion was calculated using the Wilson / Brown method. An A*01:01 monospecific pattern of reactivity was observed significantly more often than expected (34.0% vs. 14.3%, p=0.0004).

[0027] FIGs. 10A-10C show rmAb binding to HLA cytometric bead arrays. Binding of A*01 :01-specific rmAbs to microbeads displaying 21 individual HLA-A proteins. Binding to each HLA-A protein reported as net gMFI. Rows annotated with lineage IDs and rmAb names. In FIG. 10A, 17 rmAbs, derived from 8 distinct clonal lineages, each displayed an A*01 :01-monospecific reactivity pattern (A1+ pattern). In FIG. 10B, 5 rmAbs, derived from 2 distinct clonal lineages, each displayed an A*01:01+A*29:02+A36:01+A*80:01+ reactivity pattern (A1+A29+A36+A80+ pattern). In FIG. 10C, 10 rmAbs, derived from a single clonal lineage (Linl) displayed multiple reactivity patterns. However, 90% of the Linl rmAbs bound (≥40,000 net gMFI) at least six alleles A*01 :01+A*03:01+A*l l:01+A*24:02+A*36:01+A*80:01+ (A1+A3+A11+A24+A36+A80+ consensus reactivity pattern).

[0028] FIGs. 11A-11N show the immunodominant alloHLA-specific Abs exhibit focused recognition of the HLA-A*01 :01 crown region. FIGs. 11 A-l 1C show HLA-A reactivity profiles showing binding of kidney Bmem cell derived rmAbs E07 (FIG. 11 A), L02 (FIG. 11B) and M07 (FIG. 11C) to microbeads displaying 21 individual HLA-A proteins. Data reported as net gMFI of the average of 2 technical replicates of rmAbs that were diluted from 10 pg / mL to 5 ng / mL. Donor allele (“d”) and self-alleles (“s”) indicated. The HLA reactivity profile for each rmAb is defined as all HLA-A alleles (bolded) with a net gMFI ≥40,000 following incubation with the rmAbs at 10 pg / mL. FIG. 11D- 11I show characterization of HLA-A*01 :01 epitopic AA residues bound by rmAbs E07, L02 and M07 derived from recipient N006 kidney Bmem cells. FIGs. 11D, 11F, 11H highlight the A*01 :01 a-chain AA residues (top row, residues numbered 1-274) in each structurally defined HLA-A*01:01 epitope bound by rmAb E07 (FIG. 11D, n=20 residues), L02 (FIG. 11F, n=21 residues) and M07 (FIG. 11H, n=22 residues). The epitopic HLA-A*01:01 residues are aligned with other HLA-A alleles, including self-alleles A*24:02 and A*30:01 and third-party HLA alleles also bound by E07, L02 or M07 (see FIGs. 11 A-l 1C). Identical residues between the A*01 :01 donor allele and both self-alleles (0 mismatches or 0MM are shown as dots (“.”). Residues mismatched between self, donor or third-party alleles are indicated with the one letter AA code. Residues double-mismatched against both self-alleles (2MM) or single-mismatched (1MM) against one self-allele (A* 24: 02 or A*30:01) indicated. HLA alleles bound by the rmAb (gMFI ≥40,000 at 10 pg / mL) are bolded. Heatmaps in panels E (rmAb E07), G (rmAb L02) and I (rmAb M07) depict the A*01:01 epitopic residues involved in hydrogen bonds with the rmAbs (left heatmap), the 0MM, 1MM or 2MM A*01:01 epitopic residues (middle heatmap) and the amount of surface area of each epitopic residue that is buried by rmAb binding (right heatmap). FIG. I ll shows depiction of the three epitopic footprints of E07, L02, and M07 bound to A*01:01. Regions of overlap between epitopes indicated. FIG. 11K shows location of the four 2MM residues (A76, V150, V158, R163) within the A*01:01 epitopes bound by rmAbs E07, L02, or M07. Peptide, 2MM residues and AH-PBG region are shown. FIGs. 11L and 11M are pie charts depicting the location of 2MM and 1MM plus 2MM (1+MM) residues within the HLA epitopes bound by E07, L02, and M07, comparing epitopic MMs (left pie chart for each figure) versus non-epitopic MMs (right pie chart for each figure). Each pie chart indicates the proportion of MMs contained within the AH-PBG region (grey) or contained outside the AH-PBG region (black). FIG. 11L depicts 2MM residues with ≥50 A2 of solvent-accessible surface area (n=7). FIG. 11M depicts all 1MM and 2MM residues (1+MM) with ≥50A2 of solvent-accessible surface area (n=14). FIG. 1 IN shows the distance of solvent-accessible 2MM residues (left, n=7) or 1+MM residues (right, n=14) from the center of the PBG in A*01:01 (see Figure 8A for all 2MM residues).

[0029] Residues contained in the HLA epitopes bound by E07, L02, or M07 (first bar in both graphs) and residues outside of the HLA epitopes recognized by these 3 rmAbs (second bar in both graphs). Statistical analysis for FIGs. 11L and 11M performed using Fisher’s exact test. Comparisons in FIG. 11N used an unpaired t-test. All distributions passed normality (Shapiro-Wilk test).

[0030] FIGs. 12A-12G show the physicochemical, structural and topographic features of HLA A*01:01 epitopes. FIG. 12A and 12B show physiochemical analyses of solvent- accessible (≥50 A2) AA residues that were mismatched (1MM or 2MM) between donor HLA-A*01 :01 and recipient (N006) self-HLA alleles. In FIG. 12A, residues that were either 1MM or 2MM were grouped together based on whether residues were located within (epitopic, first bar in all graphs, n=9 residues) or were not present in the epitope footprint (non-epitopic, second bar in all graphs, n=5 residues) recognized by E07, L02, and / or M07. In FIG. 12B, 2MM residues were grouped together based on whether residues were located within (epitopic, first bar in all graphs, n=4 residues) or were not present in (non-epitopic, second bar in all graphs, n=3 residues) the epitope footprint recognized by E07, L02, and / or M07. Measurements of protrusion index, hydrophilicity, isoelectric point, hydrophobic mismatch, isoelectric point mismatch and dissimilarity are shown for n=14 1MM or 2MM residues (FIG. 12A) and n=7 2MM residues (FIG. 12B). For hydrophobicity mismatch, isoelectric point mismatch, and dissimilarity, the absolute difference of those physicochemical parameters was used to calculate a mismatch score for each residue. See Methods in examples for details. FIG. 12C and 12D are scatterplots showing the topography and solvent accessibility of mismatched residues in the HLA A*01 :01 epitopes recognized by E07, L02, or M07. Data displayed as solvent accessible surface area (x-axis, in A2) of each residue in A*01 :01 (n=274) and the distance of each residue from the center of the peptide- binding groove (y-axis, in A). FIG. 12C displays 2MM epitopic residues (n=4, dark grey), 2MM non-epitopic residues (n=9, black) and all other OMM or 1MM residues (n=261, white open circles). FIG. 12D displays all 1+MM epitopic residues (n=16, dark grey), 1+MM non- epitopic residues (n=17, white open circles) and all other OMM residues (n=241, grey). FIG. 12E and 12F are pie charts depicting the location of mismatched (MM) residues within the HLA epitopes bound by the rmAbs E07, L02, and M07, comparing epitopic MM residues (left pie chart) versus non-epitopic MMs (right pie chart). Each pie chart indicates the proportion of MMs contained within the AH-PBG region (left circle for each figure) or localized outside the AH-PBG region (right circle for each figure). FIG. 12E depicts 2MM residues with ≥30 A2 of solvent-accessible surface area (n=7). FIG. 12F depicts all 1+MM residues with ≥30 A2 of solvent-accessible surface area (n=19). FIG. 12G shows the distance of 2MM residues (left, n=7) or 1MM plus 2MM residues (right, n=I9) with ≥30 A2 of solvent-accessible surface area from the center of the peptide binding groove in A* 01:01. Residues contained in the HLA epitopes bound by E07, L02, or M07 (first bar for each graph) and residues outside of the HLA epitopes recognized by these 3 rmAbs (second bar for each graph). Data for FIGs. 12A and 12B analyzed using unpaired t-test (normal distribution) or Mann- Whitney test (non-normal distribution); normality testing performed using Shapiro-Wilk test. Statistical analysis (FIG. 12E and 12F) performed using Fisher’s exact test. Comparisons in (FIG. 12G) used an unpaired t-test; all distributions passed normality (Shapiro-Wilk test).

[0031] FIG. 13 shows affinity of binding by rmAb E07 to single residue mutants of HLA- A*01 :01 and examines solvent accessible mismatched residues located in the HLA crown. FIG. 13A shows binding to A*01:01 wild-type (WT) protein by the pan anti-human HLA Class I-reactive Ab W6 / 32 (mouse anti-human HLA / 2m reactive Ab). Data shown as decreasing concentration of W6 / 32 (x-axis) and gMFI for binding to A* 01 :01 microbeads (y- axis). FIG. 13B shows distance (A) between the heavy atoms in E07 IgH and IgL chains and the 1MM A*01 :01 residues G56 and Q62. Solid lines show distances to heavy atoms in E07. Peptide in peptide binding groove shown and labeled. FIGs. 13C and 13D show binding affinities to A*01:01 WT protein and single residue mutants by the rrnAbs E07 and L02. A*01 :01 mutants included reversions of three mismatched residues (V158, R163, D166) within the A*01:01 epitope recognized by E07 to the residue expressed by recipient N006 self-alleles (V158A, R163T, D166E). KD values were measured using L-SPR. Lines indicate median of three technical replicates. Dotted line indicates limit of detection (KD 4.3 pM) for the assay. FIG. 13E shows comparison of solvent-accessible 2MM residues contained within the AH-PBG region for a representative cohort of 247 HLA-A genotyped subjects who did not express A*01:01 as a self-allele. For each subject, the number of solvent-accessible (≥50 A2) 2MM residues was determined by comparing the AA sequences of their self-HLA alleles to the non-self A*01:01. Solvent accessible 2MM residues were then classified as “covered” by the E07 and L02 rmAbs if the residue was contained within the E07 or L02 epitope. FIG. 13F shows comparison of solvent-accessible (≥50 A2) 2MM residues contained within the AH-PBG region for 11 subjects who received an HLA-mismatched A*01:01 solid organ transplant and subsequently developed anti-A*01:01 DSA. For each subject, the number of solvent-accessible 2MM residues was determined by comparing the AA sequences of their self-HLA alleles to the non-self A*01 :01 expressed by the allograft. Solvent accessible 2MM residues were classified as “covered” by the E07 and L02 rmAbs if the 2MM was contained within the E07 or L02 epitope.

[0032] FIGs. 14A-0 show convergent recognition of the HLA-A*01:01 crown-localized epitopes by multiple distinct B cell clonal lineages and identifies critical amino acid residues required for binding of rmAb E07. FIG. 14A shows chimeric mouse / human rmAbs cE07 and cL02 block binding of A*01 :01 specific rmAbs derived from 10 independent B cell clonal lineages. HLA-A*01 :01 microbeads were blocked with chimeric A*01 :01-specific cE07 and / or cL02 rmAbs, then stained with human A*01 :01-specific test human rmAbs (n=22, Figure 10A-10B) that were subsequently detected with fluorochrome-labeled anti-human IgG. Data reported as percent inhibition of test rmAb binding (mean of ≥2 technical replicates) and are representative of 2 independent experiments. Test human A*01:01- specific rmAbs subdivided based on clonal lineage and HLA reactivity patterns. W6 / 32, a murine Ab specific for human 2m, included as a negative control for inhibition. FIGs. 14B and 14C show the comparison between HLA binding reactivity pattern of test rmAbs and inhibition of test rm Ab binding to A*01:01 mediated by chimeric rmAbs cE07 or cL02. Data shown as % inhibition with test rmAbs grouped by HLA binding patterns. FIGs. 14D-14G show binding to HLA-A*01:01 wild-type (WT) protein and single residue mutants of A*01 :01 (V158A, R163T, D166E) by rmAbs E07 (FIG. 14D) and L02 (FIG. 14E). Data shown as increasing concentrations of the rmAbs (x-axis) and percent of maximum gMFI for binding to A*01:01 (y-axis). Binding curves were fit to each rmAb:protein combination and used for EC50 calculations ((FIG. 14F-14G), see Methods in examples for details). Data shown for three independent experiments for each rmAb:protein combination, with lines indicating mean with standard deviation. FIGs. 14H-14J show interactions of rmAb E07 with 2MM and 1MM residues in A*01:01. Top cartoons show interactions between residues in the E07 IgVH domain (heavy chain [HC]) or IgVL domain (light chain [LC]) and A*01:01 (alpha helix cartoon) with selected mismatched AA residues in A*01 :01 (stick model with selected amino acids called out), including 2MM VI 58 (FIG. 14H), 2MM R163 (FIG. 141), and 1MM DI 66 (FIG. 14 J). Bottom cartoons show reduced contact between E07 and HLA structures harboring A*24:02 / A*30:01 self-reversion mutations V158A (FIG. 14H), R163T (FIG. 141), or D166E (FIG. 14 J). Polar interactions noted by dashed lines. FIG. 14K is a heatmap showing EC50 values for 22 test rmAbs binding to WT and mutant A*01 :01 microbeads. Data shown as mean of three independent experiments for each rmAb:protein combination. Test rmAbs grouped by clonal lineage and HLA reactivity pattern. FIG. 14L is a heatmap showing relative reduction in binding to A*01 :01 mutants by 22 A*01 :01 specific rmAbs. Data reported as the fold change in EC50 values (mutant EC50 / WT EC50) for each rmAb and shown as mean of three independent experiments for each rmAb:protein combination. Test rmAbs grouped by clonal lineage and HLA reactivity pattern. FIGs. 14M- 140 show comparison of test rmAb binding to A* 01 :01 mutants to chimeric cE07 rmAb mediated inhibition of test rmAb binding to A*01:01. Binding of test rmAbs to A*01:01 mutants shown as fold-change in EC50 values (mutant EC50 / WT EC50). Test rmAbs grouped based upon their percent inhibition (≥50% or <50%) by cE07. Test rmAbs are distinguished in grey scale based upon anti-HLA binding patterns. Statistical analysis (FIGs. 14B-14C, 14M-14O) performed using a Mann- Whitney test. Nonlinear regression with a sigmoidal four-parameter logistic curve (FIGs. 14D-14G, 14K) used to fit binding for each rmAb:protein combination and calculate EC50 values. A*01:01 mutant EC50 values were compared against the WT EC50 values with Brown-Forsythe and Welch ANOVA tests (FIGs. 14F, 14G, 14K) with Dunnett’s T3 multiple comparisons test. In FIG. 14K, statistical significance is indicated with asterisks: * P<0.05, ** P<0.01, *** P<0.001. FIGs. 15A-Q shows that E07 and L02 rmAbs can inhibit the binding of immunodominant antibody responses to HLA-A*01:01 across a cohort of transplant patients. FIGs. 15A-15C show chimeric mouse / human rmAbs cE07 and cL02 block binding of circulating polyclonal Abs from recipient N006. Data reported as gMFI (mean plus standard deviation for ≥2 technical replicates per condition) of binding by total plasma polyclonal Abs (FIG. 15A) or affinity-purified A*01:01 binding appAbs (FIG. 15B) to A*01 :01 coated microbeads. Data in (FIG. 15C) reported as percent inhibition of binding and represent the mean of at least two technical replicates. W6 / 32, a murine Ab specific for human class I HLA, included as a negative control for inhibition. FIGs. 15D and 15E show binding to wild type (WT) A*01 :01 and mutant A*01:01 (V158A, R163T, D166E) by appAbs from recipient N006. Data are shown as binding activity (arbitrary units, y-axis) for each mutant HLA-A in FIG. 15D and the percentage of WT binding activity in FIG. 15E. Binding activity was calculated by interpolating binding activity using a standard curve of W6 / 32 binding for each mutant (FIG. 13 A). Data representative of three independent experiments for each appAb:protein combination. FIG. 15F shows HLA residues contained within the structurally- defined HLA epitopes recognized by E07 and L02 labeled and circled and within the HLA epitopes predicted by GraphBepi and DiscopTope-3.0 (grey) displayed on the HLA-A*01:01 complex with a-chain (black) and peptide (offwhite) residues. FIG. 15G are pie charts comparing structurally defined and computationally predicted HLA-A*01 :01 epitopic residues within the 74 AA A*01:01 AH-PBG region. Chart 1 (from left to right) shows proportion of residues located within AH-PBG relative to the total number (n=274) of HLA- A*01 :01 a-chain residues. Chart 2 shows proportion of residues within the epitopes recognized by E07 and L02 (n=41) that are localized within the AH-PBG region. Charts 3 and 4 show proportion of residues within the GraphBepi predicted epitope (n=58, pie chart 3), or the DiscoTope-3.0 predicted epitope (n=30, chart 4) that are localized within the AH- PBG region. FIG. 15H is a histogram showing frequency distribution of solvent-accessible (≥50 A2) 2MM residues that are localized within the A*01 :01 epitopes recognized by E07 and L02 rmAbs in a representative cohort (n=247) of HLA-A genotyped subjects who do not express HLA-A*01:01 as a self-allele. FIG. 151 is a heatmap depicting the HLA-A reactivity pattern of serum from 11 transplant recipients who developed a DSA response to A*01 :0I expressed by the mismatched donor allograft. Clinical HLA laboratory cytometric bead array data reported as the MFI for serum binding to 31 HLA-A alleles. Self-alleles for each subject indicated (“s”) and subjects homozygous for a given allele indicated with “ss.” For a single subject (recipient 11), one of the self-alleles (A*66:03) was not included in the HLA cytometric bead array. FIG. 15J shows localization of solvent-accessible (≥50 A2) 2MM A*01 :01 residues identified in 11 DSA+ transplant recipients receiving a A*01 :01 mismatched allograft. Data shown as the percent of recipients (y axis) with a 2MM residue located at positions 1 to 274 of the A*01 :01 a-chain (x axis). 2MM residues within the AH- PBG region are highlighted with a box. FIG. 15K is a histogram showing frequency distribution and location of solvent-accessible (≥50 A2) 2MM residues within the A*01 :01 epitopes recognized by E07 and L02 rmAbs in 11 transplant recipients who received a mismatched A*01:01 allograft. The total number of solvent-accessible 2MM residues located within the AH-PBG region was determined for each subject and the percentage of these 2MM residues that were contained within the A*01:01 epitopes bound by E07 and L02 was determined. FIGs. 15L-15M show chimeric mouse / human rmAbs cE07 and cL02, derived from Bmem isolated from the kidney of recipient N006, block binding to A* 01 :01 by polyclonal Abs found in serum samples isolated from 11 DSA+ transplant recipients who received a A*01:01 mismatched allograft. Data reported as percent inhibition of serum binding and shown as the mean two independent experiments with at least two technical replicates / experiment. W6 / 32, a murine Ab specific for human class I HL A, included as a negative control for inhibition. FIG. 15N shows classification of HLA a-chain residues 158, 163, and 166 for 11 DSA+ transplant recipients who received a mismatched A*01:01 allograft. The residues present in A*01:01 at positions 158, 163 and 166 were compared to the residues expressed by each subject’s self-HLA-A alleles at the same position. Residues were classified as 2MM, 1MM or OMM between A*01:01 and the self-HLA alleles. FIGs. 15O-15P show binding to WT A*01:01 and mutant A*01:01 (V158A, R163T, D166E) by serum samples from 11 DSA+ transplant recipients who received a A*01 :01 mismatched allograft. Data shown as binding activity (arbitrary units, AU) for each WT or mutant A*01 :01 protein (FIG. 150) or as percentage of WT binding activity in (FIG. 15P). Binding activity was calculated by interpolating binding activity using a standard curve of W6 / 32 binding for each mutant (see Methods for details). Data are shown as mean values from three independent experiments for each subjectprotein combination. FIG. 15Q shows correlation between loss of binding to HLA-A*01 :01 mutants (V158A, R163T, D166E) and chimeric cE07-dependent binding inhibition in serum samples from 11 DSA+ transplant recipients who received a A*01:01 mismatched allograft. Data shown as scatterplots with x-axis displaying % inhibition by chimeric cE07 (top plot) or cL02 (bottom plot) rmAbs and y-axis displaying percent binding (mutant binding AU / WT binding AU x 100). Values in FIGs.

[0033] 15A-B were compared against the non-blocking condition using the Kruskal-Wallis test with Dunn’s multiple comparisons test. Values in FIG. 15D were compared against WT binding using the Kruskal-Wallis test with Dunn’s multiple comparisons test. Proportions in FIG. 15G were compared using Fisher’s exact test. Group ranks in FIG. 15L were compared using the Friedman test with Dunn’s multiple comparisons test against the W6 / 32 condition. Group ranks in FIG. 150 were compared using the Friedman test with Dunn’s multiple comparisons test against the WT protein. Statistical analysis in FIG. 15Q performed using a nonparametric Spearman correlation with a two-tailed P value.

[0034] FIG. 16 shows the binding footprint and interactions of rmAb 122 to HLA-A*01:01. FIG. 16A shows structures showing IgH (HC) and IgL (LC) chains of kidney Bmem cell derived A*01:01-specific rmAb 122 bound to HLA-A*01:01 [HLA-A*01:01 a-chain and peptide indicated]. The number of A*01 :01 a-chain AA residues contained within each epitope and the amount of A*01 :01 a-chain surface area buried upon binding of 122 are indicated. FIG. 16B shows the epitope covered by 122 is shown upon the HLA A*01 :0 a- chain. Epitopic residues are shown shaded in black. The bound peptide is indicated. FIG. 16C shows polar interactions of rmAb 122 with residues in A*01:01. The cartoon shows interactions between residues in the 122 IgVL domain (light chain [LC]) and A*01:01 (alpha helix cartoon) with selected AA residues in A*01:01 (sticks). Polar interactions are noted by dashed lines.

[0035] DETAILED DESCRIPTION

[0036] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

[0037] Antibodies produced by terminally differentiated antibody secreting cells can play critical roles in protection from infection following pathogen exposure. However, antibody secreting cells and antibodies are pathogenic when directed against the wrong target, as is the case for many autoimmune diseases and HLA-mismatched organ transplantation. To date, the understanding of how mismatched HLA alleles are recognized by B cells and antibodies has been limited despite the fact that HLA molecules are a major target of the alloimmune response in solid organ transplantation. The development of DS A in previously nonsensitized recipients occurs in 7% of transplant recipients by 5 years and in nearly 20% by 10 years. Risk factors for de novo DSA responses include African American ancestry, HLA mismatches at the DQ locus, early episodes of cell-mediated rejection, and pretransplant sensitization to other donor antigens. The development of DSA in recipients of any solid organ decreases graft survival, with 40% of DSA+ patients losing their renal allografts within 5 years, whereas more than 80% of patients without DSA retain their allograft longer than 5 years. Ultimately, most late allograft failures are due to DSA-mediated chronic rejection.

[0038] Antibody secreting cells can be derived from either naive or memory B cell subsets and can develop in a germinal center dependent or independent manner. Whether the potentially pathogenic antibody secreting cells are derived from particular subpopulations within the naive or memory B cell compartment is unknown. A specialized subset of memory B cells, referred to as effector memory B cells, that are transcriptionally and metabolically poised to rapidly differentiate into antibody secreting cells, were characterized. Interestingly, these effector memory B cells do not express the follicle homing chemokine receptor, CXCR5, and instead express chemokine receptors like CXCR3, that can direct the cells to inflamed tissues and barrier sites, like the lung. Thus, effector memory B cells have the potential to provide a rapid localized antibody response following infection but also may contribute to organ destruction in chronic inflammatory diseases.

[0039] In kidney transplant recipients, donor specific antibodies (DSAs) directed against the donor’s Major Histocompatibility Complex or HLA proteins contribute to antibody-mediated rejection (AMR). Pre-formed DSA, which is present at the time of transplant, can cause acute AMR while de novo DSA, which is elicited months to years after transplantation, can cause chronic AMR. Both conditions are difficult to treat and are associated with organ failure. Given the importance of DSA and AMR in long-term outcomes for transplant recipients, HLA alloantigens and their surface features that are recognized by B cells and antibodies were analyzed and the epitope :paratope interface of the DSA and HLA were identified. Surprisingly, the footprints of the HLA epitopes from a variety of recombinant monoclonal were fairly consistent and limited to a few epitopes. Even more surprisingly, similar epitopes were recognized by DSA in multiple transplant recipients. Thus, one or more antibodies or modified antibodies that bind amino acids within the face of the HLA molecules are useful across multiple subjects. Compositions

[0040] The antibodies or modified antibodies, including combinations of the antibodies and modified antibodies, disclosed herein may block binding to at least 50%, optionally to at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the amino acids within the face of the HLA antigen A* 01 or A* 01 :01. As used herein, the face of the HLA antigen A*01 or A*01 :01 is composed of the amino acids contained in the two alpha helices that frame the HLA peptide-binding groove on the surface of the HLA protein in its three- dimensional confirmation. As used herein, blocking binding means preventing another molecule, for example an immune cell or another antibody, from binding to a particular amino acid. Such blocking does not necessarily suggest that the blocking antibody or modified antibody blocks biding to the same amino acid or amino acids to which the blocking antibody binding. Rather, the blocking antibody may by steric hinderance block binding to other amino acid residues that are located near the binding site of the blocking antibody or modified antibody. As noted throughout, the antibodies provided herein can be used in combination, for example, an antibody provided herein can be used in combination with a modified antibody. Thus, combinations of unmodified, modified and unmodified and modified antibodies can be used.

[0041] Optionally, the antibodies or modified antibodies alone or in combination disclosed herein block binding to at least two or more amino acids within the face of HLA A*01, selected from the group consisting of amino acid residues 15, 16, 54-56, 58, 62, 69, 70, 72, 73, 75-77, 79, 80, 82-84, 86-89, 106, 108, 109, 127, 129-138, 141, 144-146, 148-151, 153- 155, 157, 158, 161-163, 166, 169, 170, 173 numbered relative to SEQ ID NO: 19. In some cases, blocking binding to a certain subset of these amino acid residues will be relevant and, in other cases, other amino acid residues will be more relevant given the specific variations between subjects. In some cases, blocking binding to only a subset is needed to prevent or reduce an immune response, and, in other cases, blocking binding to all or nearly all of the amino acid residues will be needed to prevent or reduce an immune response. In some instances, one antibody or modified antibody will be sufficient, whereas, in other instances, multiple antibodies will be needed to achieve the desired effect.

[0042] Optionally, the antibodies or modified antibodies disclosed herein block binding to at least 50%, optionally to at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the donor specific antibodies to HLA A*01 or HLA A*01 :01 in a subject with donor specific antibodies. As used herein, donor specific antibodies refers to antibodies present before or after a transplant in an organ transplant recipient or potential organ transplant recipient which react or have the potential to react to the donor’s HLA antigens. In some cases, blocking binding to a 50% of the donor specific antibodies will be sufficient to reduce or avoid an immune response (including antibody mediated rejection), whereas in certain cases blocking binding to more than 50% of the donor specific antibodies is necessary (including, e.g., blocking binding to all or least 85%, 90%, 95%, or 99%) will be necessary. In some instances, one antibody or modified antibody will be sufficient, whereas, in other instances, multiple antibodies will be needed to achieve the desired effect. One of skill in the art can make the determination based on the binding patterns of a subject’s donor specific antibodies in a biological sample (e.g., a blood sample) and / or the immune state of the subject.

[0043] The HLA system includes a complex of genes on chromosome 6 in humans which encode cell-surface proteins responsible for regulation of the immune system. HLA proteins are divided into classes based on the allele by which they are encoded: Class 1 (A, B, and C) and Class II (DP, DM, DO, DQ, and DR). HLA proteins present peptide antigens to the immune system. Class I HLAs present peptides from inside the cell, while Class II HLAs present peptides derived from external antigens. HLA alleles have either a two- or four-digit designation depending on the level of granularity identified. For example, HLA A*01 can encompass HLA A*01:01, A*01 :02, and A*01:03. As used herein, HLA A*01 refers to each of HLA A*01:01, A*01:02, and A*01:03.

[0044] The antibodies or modified antibodies as described herein optionally bind to more than one HLA allele. For example, the antibodies or modified antibodies could bind one or more Class A*01 alleles. Additionally, the antibodies or modified antibodies could bind one more alleles of other HLA classes.

[0045] As used herein, antibodies refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0046] The antibodies or modified antibodies described herein may IgG, IgM, IgA, IgD and IgE antibodies, or a portion thereof. Optionally, the antibodies or modified antibodies described herein are IgGs or any subclass thereof, e.g., IgGl, IgG2, IgG3, or IgG4 antibodies, or modified versions (e.g., portions) thereof. Naturally occurring immunoglobulins have a common core structure in which two identical light chains and two identical heavy chains form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. The terms variable light chain (VL) and variable heavy chain (VH) refer to variable regions of the light and heavy chains respectively. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. The CDR region may further be designated by its position on either the heavy or light chain variable regions, for example CDRH1, CDRH2, or CDRH3 or CDRL1, CDRL2, or CDRL3, respectively.

[0047] The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by the IMGT Scientific Chart (See https: / / www.imgt.org / IMGTScientificChart / ).

[0048] The one or more antibodies or modified antibodies provided herein may be full length antibodies or modified antibodies. Modified antibodies, used herein, refers to fragments of the antibodies that retain the abilities (e.g., binding and / or blocking) of the full-length antibodies provided herein. For example, the ability to bind an HLA antigen A*01 or A*01 :01 such that the bound one or more antibodies block binding to at least 50% of the amino acids within a face of HLA A*01 or A*01:01.

[0049] The one or more antibodies or modified antibodies provided herein may comprise one or more single chain antibodies or portions thereof. Optionally, single chain antibody is a single chain variable fragment (scFvs). Optionally, the one or more antibodies or modified antibodies provided herein comprise one or more Fab’ fragments. Optionally, the one or more antibodies or modified antibodies provided herein comprise one or more F(ab')2 fragments.

[0050] Antibody fragments optionally include additional components, such as one or more peptide linkers. Such fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity. (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)). Thus, the term modified antibody, as used herein, includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries. (See e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0051] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used. (See e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). Monoclonal antibodies refer to antibodies derived from a single B cell clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens. (See, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0052] The one or more antibodies or modified antibodies provided herein may comprise one or more chimeric antibodies or portions thereof. As used herein, chimeric antibodies refers to an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0053] The one or more antibodies or modified antibodies provided herein may comprise one or more bispecific antibodies. As used herein, bispecific antibodies possess dual or multiple antigen or epitope specificities. Bispecific antibodies described herein may block binding to different amino acids within the face of HLA A*01 or A*01 :01.

[0054] The antibodies or modified antibodies described herein may be human. As used herein, human refers to antibodies or modified antibodies that are fully human (i.e., derived from a human or an animal genetically engineered to produce human antibodies) or that are humanized. A humanized antibody is an antibody from a non-human species that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g. , Morrison et al. , Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994). The humanized antibodies or modified antibodies described herein may be derived from any non-human species, for example, mouse, non-human primates, rabbit, dog, or chicken.

[0055] The antibodies provided herein can contain modifications in one or more regions of the antibody. By way of example, the antibodies provided herein can include one or more modifications in the Fc region of the antibody. The Fc region of an antibody is the tail region of an antibody that can interact with cell surface receptors called Fc receptors and other proteins. Modifications to the Fc region of an antibody are known and include those listed in Table 1. The modifications are based on the EU numbering system, which is based on IMGT and included in the following IMGT scientific chart: https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. The Fc region modifications (EU Numbering) can be used alone or in combination and are selected from the group consisting of N297A, N297Q, L234A, L235A, G327A, L235A, P29G, P29A, L234, G237A, S267K, P329A, G237, D265A, L234S, L235T, G236 R, G236R, L328R , P329A , L234F, L235E, P331S, L235Q, K322Q, P238S, H268A, A330S, E233P, L234V, DG236,

[0056] A327G, L235G, G236R, M252Y, S254T, and T256E. These modifications can be used, in some cases, to render the antibody more suitable for therapeutic use.

[0057] Table 1. Reported IgGl Fc mutants to enhance therapeutic use of antibodies.

[0058] References for Table 1.

[0059] 1. Bolt, S., et al., The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties. Eur J Immunol, 1993. 23(2): p. 403-11. 2. Lund, J., et al., Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol, 1991. 147(8): p. 2657-62.

[0060] 3. Wilkinson, I., et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One, 2021. 16(12): p. e0260954.

[0061] 4. Horton, H.M., et al., Fc-engineered anti-CD40 antibody enhances multiple effector functions and exhibits potent in vitro and in vivo antitumor activity against hematologic malignancies. Blood, 2010. 116(16): p. 3004-12.

[0062] 5. Oganesyan, V., et al., Structural characterization of a human Fc fragment engineered for lack of effector functions. Acta Crystallogr D Biol Crystallogr, 2008. 64(Pt 6): p. 700-4.

[0063] 6. Borrok, M.J., et al., An "Fc-Silenced" IgGl Format With Extended Half-Life Designed for Improved Stability. J Pharm Sei, 2017. 106(4): p. 1008-1017.

[0064] 7. Tam, S.H., et al., Functional, Biophysical, and Structural Characterization of Human IgGl and IgG4 Fc Variants with Ablated Immune Functionality. Antibodies (Basel), 2017. 6(3).

[0065] 8. Geuijen, C.A.W., et al., Unbiased Combinatorial Screening Identifies a Bispecific IgGl that Potently Inhibits HER3 Signaling via HER2-Guided Ligand Blockade. Cancer Cell, 2018. 33(5): p. 922-936 elO.

[0066] 9. DallAcqua, W.F., P.A. Kiener, and H. Wu, Properties of human IgGls engineered for enhanced binding to the neonatal Fc receptor (FcRn). J Biol Chem, 2006. 281(33): p. 23514- 24.

[0067] 10. Jeffrey L. Bennett (2020) Compositions and methods for the treatment of neuromyelitis optica. US Patent No. 11,390,667

[0068] 11. Brian Walter Granda (2023) Antibody Fc Variants. US Patent Application Publication No. 2024 / 0002509 Al

[0069] 12. Leonard Presta (2000) Polypeptide variants with altered effector function US Patent No. 6,737,056

[0070] 13. U.S. Publication No. 2022 / 0289803 by Liu et al.

[0071] As used herein, amino acids refer to the typically encountered twenty amino acids which make up polypeptides. The terms peptide and polypeptide are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The term protein as used herein refers to either a polypeptide, a dimer (i.e., two polypeptides), or multimer (i.e., three or more polypeptides) of single chain polypeptides that may be the same or different polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.

[0072] The term epitope, as used herein, means a component of an antigen (such as an HLA A*01 or A*01:01 antigen) capable of being bound by an antibody or modified antibody. Such components optionally comprise one or more contiguous amino acid residues and / or one or more non-contiguous amino acid residues, depending on the three-dimensional conformation of the antigen. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations.

[0073] Optionally, the one or more antibodies or modified antibodies provided herein comprise one or more of (a) a first antibody or portion thereof, wherein the first antibody or portion thereof comprising CDRs having the amino acid sequences of SEQ ID NOs: 1-6; (b) a second antibody or portion thereof, wherein the second antibody or portion thereof comprising CDRs having the amino acid sequences of SEQ ID NOs: 7-12; and (c) a third antibody of portion thereof, wherein the third antibody or portion thereof comprising CDRs having the amino acid sequences of SEQ ID NOs: 13-18. The CDRs form the paratope that binds the epitope of the HLA.

[0074] Optionally, the one or more antibodies or modified antibodies provided herein comprise a heavy chain variable region comprising (i) a CDRH1 with an amino acid sequence comprising SEQ ID NO:1, (ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO:2, and (iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:3; and (b) a light chain variable region comprising (i) a CDRL1 with an amino acid sequence comprising SEQ ID NO:4, (ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO:5, and (iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO:6. Optionally, the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 54-56, 58, 62, 106, 108, 109, 129, 131, 154, 155, 157, 158, 161-163, 166, 169, and 170 within the face of the human leukocyte antigen A*01:01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19. Optionally, the one or more antibodies or modified antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:26. Optionally, the one or more antibodies or modified antibodies comprise a light chain having the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:27. An example of this antibody is designated herein as the E07 antibody.

[0075] Optionally, the one or more antibodies or modified antibodies provided herein comprise (a) a heavy chain variable region comprising (i) a CDRH1 with an amino acid sequence comprising SEQ ID NO:7, (ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO:8, and (iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:9; and (b) a light chain variable region comprising (i) a CDRL1 with an amino acid sequence comprising SEQ ID NO: 10, (ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO:11, and (iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO: 12. Optionally, the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 15, 16, 69, 70, 72, 73, 75-77, 79, 80, 82-84, 86-89, 146, 149, and 150 within the face of the human leukocyte antigen A*01 :01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19. Optionally, the one or more antibodies or modified antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:28. Optionally, the one or more antibodies or modified antibodies comprise a light chain having the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:29. An example of this antibody is designated herein as antibody L02.

[0076] Optionally, the one or more antibodies or modified antibodies provided herein comprise (a) a heavy chain variable region comprising (i) a CDRH1 with an amino acid sequence comprising SEQ ID NO: 13, (ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO:14, and (iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:15; and (b) a light chain variable region comprising (i) a CDRL1 with an amino acid sequence comprising SEQ ID NO: 16, (ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO: 17, and (iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO: 18. Optionally, the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 127, 129, 130, 131-138, 141, 144-146, 148-151, 153, 154, and 157 within the face of the human leukocyte antigen A*01 :01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19. Optionally, the one or more antibodies or modified antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:30. Optionally, the one or more antibodies or modified antibodies comprise a light chain having the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:31. An example of this antibody is designated herein as antibody M07.

[0077] Optionally, the one or more antibodies or modified antibodies provided herein comprise a heavy chain variable region comprising (i) a CDRH1 with an amino acid sequence comprising SEQ ID NO:312, (ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO:313, and (iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:314; and (b) a light chain variable region comprising (i) a CDRL1 with an amino acid sequence comprising SEQ ID NO:315, (ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO:316, and (iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO:317. Optionally, the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 108, 109, 129, 131, 154, 157, 158, 161-163, 166, 169, 170, and 173 within the face of the human leukocyte antigen A*01:01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19. Optionally, the one or more antibodies or modified antibodies comprise a heavy chain having the amino acid sequence of SEQ ID NO:318 or SEQ ID NO:418. Optionally, the one or more antibodies or modified antibodies comprise a light chain having the amino acid sequence of SEQ ID NO:319 or SEQ ID NO:419. An example of this antibody is designated herein as the 122 antibody.

[0078] Other antibodies similar to those designated E07, L02, M07 and 122 can be used. E07, L02, M07 and 122 are used throughout as examples. Optionally, the one or more antibodies or modified antibodies provided herein comprise the sequence of those described in Table 2 below.

[0079] Table 2. Additional Antibodies

[0080] Nucleic Acids

[0081] Also provided herein are one or more nucleic acids encoding any of the one or more antibodies or modified antibodies provided herein. Also provided are nucleic acids that encode a portion of the antibodies described herein. The encoded portions are optionally CDRs or variable regions.

[0082] As used throughout, the terms nucleic acid, nucleic acid sequence, oligonucleotide, nucleotides, or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-m ethyl phosphoramidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0083] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0084] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Unless otherwise indicated, a particular polynucleotide or nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0085] Compositions

[0086] Also provided herein are compositions comprising the one or more antibodies or modified antibodies provided herein and an acceptable buffer or excipient. Optionally, the composition is a pharmaceutical composition comprising the one or more antibodies or modified antibodies provided herein and an acceptable excipient. Additionally, such compositions are optionally formulated for screening rather than for administration to a subject.

[0087] An acceptable composition may comprise, for example, a diluent, solubilizer, emulsifier, and / or preservative to be used with the methods disclosed herein. Optionally, the buffer(s) or excipient(s) used may include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; and / or pharmaceutical adjuvants. (See for example, Allen (2021) Remington - The Science and Practice of Pharmacy, 23d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). Optionally, the excipient(s) are for intravenous administration, subcutaneous administration, or intramuscular administration. The optimal composition can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. (See for example, Allen (2021) Remington - The Science and Practice of Pharmacy, 23d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). Optionally, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the one or more antibodies or modified antibodies.

[0088] Kits

[0089] Kits comprising the one or more antibodies or modified antibodies are provided.

[0090] The kits may be used in the methods described herein. For example, the kits can be used as a screening kit to identify and quantitate specific HLA-A proteins expressed by a test sample. For example, the kit can be used to identify whether human leukocyte antigen A*01 or A*01 :01 is expressed by cells or tissues.Optionally, the kits may be used in blocking assays to screen samples, such as a blood samples from a patient, to identify the HLA epitopes bound by the anti-HLA antibodies present in a patient sample. Optionally, the kits can further include human leukocyte antigen (HLA) proteins or portions thereof. The HLA proteins or portions thereof can be used as positive controls in using the kits in methods of screening and / or blocking assays.

[0091] Optionally, the one or more antibodies or modified antibodies provided herein will be in solution in a diluent. Optionally, the HLA proteins or portions thereof comprising one or more epitopes thereof, will be in solution in a diluent. Optionally, the one or more antibodies or modified antibodies provided herein will be attached to a solid support. Optionally, the HLA proteins or portions thereof comprising one or more epitopes thereof, will be attached to a solid support. A solid support may include, for example, mobile beads, a slide, culture dish, multiwell plate, column, chip, array, or stable beads.

[0092] In the herein provided kits, the one or more of the antibodies or modified antibodies may be labeled with an enzymatic or fluorescently label that can be used to quantitate the amount of labeled antibody that binds to a test sample, that may include for example, blood cells or tissue samples from an individual.

[0093] Optionally, the kits described further compromise one or more modified antibodies in a diluent with one or more HLA proteins or portions thereof comprising one or more epitopes thereof attached to a solid support.

[0094] The herein provided kits may also have containers containing buffer(s) and / or a container comprising a reporter-means, such as an enzymatically or fluorescently label secondary antibody that can specifically distinguish between the antibodies provided in the kit and the antibodies present in the test material.

[0095] Optionally, the kits described further comprise one or more devices for therapeutic delivery or the one or more antibodies or modified antibodies to a subject. The one or more devices for therapeutic delivery may be, for example, a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, with one or more of the containers comprising the one or more antibodies or modified antibodies provided herein. The kit may also have containers containing buffer(s) and / or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label.

[0096] The kit may have containers containing a diluent, solubilizer, emulsifier, and / or preservative to be used with the methods disclosed herein. The kit optionally contains a diluent formulated for intravenous infusion of the one or more antibodies or modified antibodies provided herein.

[0097] Optionally, the kit comprises the one or more antibodies or modified antibodies provided herein in a single unit dosage form or as separate unit doses. The dose and form of the unit dose (e.g., tablet, capsule, immediate release, delayed release, etc.) can determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. Optionally, the kit may include the one or more antibodies or modified antibodies provided herein in a single-dose administration unit. Optionally, kits containing single or multi-chambered pre-filled syringes comprising the one or more antibodies or modified antibodies are included.

[0098] Methods of Treatment

[0099] Provided herein is a method of reducing an immune response in a transplant recipient to a donor organ comprising administering to the transplant recipient the one or more antibodies or modified antibodies described herein in an amount effective to reduce the immune response in the transplant recipient as compared to a control immune response in the absence of administration of the one or more antibodies or modified antibodies. Optionally, the immune response comprises antibody mediated rejection of a transplanted organ or tissue. As noted above, the antibodies provided herein can contain modifications in one or more regions of the antibody. By way of example, the antibodies provided herein can include one or more modifications in the Fc region of the antibody. The Fc region of an antibody is the tail region of an antibody that can interact with cell surface receptors called Fc receptors and other proteins. Modifications to the Fc region of an antibody are known and include those listed in Table 1. The modifications (EU Numbering) can be used alone or in combination and are selected from the group consisting of N297A, N297Q, L234A, L235A, G327A, L235A, P29G, P29A, L234, G237A, S267K, P329A, G237, D265A, L234S, L235T, G236 R, G236R, L328R , P329A , L234F, L235E, P331S, L235Q, K322Q, P238S, H268A, A330S, E233P, L234V, DG236, A327G, L235G, G236R, M252Y, S254T, and T256E.

[0100] Administration can occur by using any number of means available in the art. Typically, the one or more antibodies or modified antibodies is administered to the subject intravascularly, subcutaneously, intramuscularly, transdermally (e.g., by a transdermal patch or a topically applied cream, ointment, or the like), orally, intrapulmonarily, transmucosally, intraperitoneally, intrauterinely, sublingually, intrathecally, intramuscularly, intraarticularly, etc. using conventional methods. In addition, the one or more antibodies or modified antibodies can be administered via injectable depot routes such as by using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0101] The one or more antibodies or modified antibodies of the present disclosure can be administered to the subject before transplantation, including near or at the time of transplantation. For example, the one or more antibodies or modified antibodies can be administered substantially simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute, or less, before transplantation) or about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, or more, before transplantation.

[0102] The one or more antibodies or modified antibodies of the present disclosure can be administered to the subject at any time after transplantation. Optionally, the one or more antibodies or modified antibodies can be administered to the subject upon clinical signs of a donor specific antibody response determined by one skilled in the art. Optionally, the one or more antibodies or modified antibodies can be administered to the subject when immunosuppressants are ineffective or unavailable. For example, upon infection when immunosuppressants are necessarily reduced or eliminated from a treatment regimen, the antibodies or modified antibodies can be administered instead of or with a reduced dosage of the immunosuppressant.

[0103] Regardless of the route of administration, the amount or schedule of administration of the one or more antibodies or modified antibodies will vary among individuals based on age, size, weight, condition to be treated, mode of administration, and the severity of the condition. One skilled in the art will realize that dosages are best optimized by the practicing physician and methods for determining dosage are described, for example in Remington’s Pharmaceutical Science, latest edition. (See for example, Guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389). A typical dose of the one or more antibodies or modified antibodies used alone might range from about 0.1-100 mg / kg (including for example, 1-50 mg / kg) of body weight when given intravenously by infusion or 5 mg-1 g (including, for example, 50-500 mg) per dose when injected subcutaneously, depending on the factors mentioned above. Doses are optionally delivered daily, on alternating days, weekly, or monthly.

[0104] Optionally, the method of reducing an immune response in a transplant recipient to a donor organ further comprises screening a biological sample from the subject to identify the one or more antibodies or modified antibodies described herein that block binding of at least 50%, optionally to at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of donor specific antibodies to human leukocyte antigen A*01 or A*01:01 in the subject.

[0105] As used throughout, a biological sample refers to a sample from the subject. The sample can be, but is not limited to, peripheral blood, plasma, serum, transplant tissue biopsies, urine, saliva, gastric secretion, feces, bone marrow specimens, primary tumors, embedded tissue sections, frozen tissue sections, cell preparations, cytological preparations, exfoliate samples (e.g., sputum), fine needle aspirations, amnion cells, fresh tissue, dry tissue, and cultured cells or tissue, ft is further contemplated that the biological sample of this invention can also be whole cells or cell organelles (e.g., nuclei). A biological sample can also include a partially purified sample or a cell culture. The sample can be in solution or on, supported by, or attached to, a substrate which facilitates detection. A substrate can be, but is not limited to, a bead, a microscope slide, a culture dish, a culture flask, a culture plate, a culture chamber, ELISA plates, as well as any other substrate that can be used for containing or supporting biological samples for analysis according to the methods of the present invention. The substrate can be of any material suitable for the purposes of this invention, such as, for example, glass, plastic, polystyrene, mica and the like. The substrates can be obtained from commercial sources or prepared according to standard procedures well known in the art.

[0106] As used throughout, by a subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. The term subject can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, pig, etc.).

[0107] The identifying step of the method of reducing an immune response in a transplant recipient can be selected from methods routine in the art. For example, the identifying step can be performed in vivo using a noninvasive medical technique such as radiography, fluoroscopy, sonography, imaging techniques such as magnetic resonance imaging, and the like. Thus, for example, the disclosed one or more antibodies or modified antibodies, can be labeled for identification in a subject using an appropriate imaging or detection modality. If, for example, the one or more antibodies or modified antibodies are radiolabeled then they can be identified using radiologic detection. Similarly, if the one or more antibodies or modified antibodies are labeled fluorescently, then they can be identified with a fluorescence detector. In vitro identification methods can be used to identify the bound one or more antibodies or modified antibodies in an ELISA, RIA, immunohistochemically, flow cytometry, FACS, IHC, FISH, proteonomic arrays, protein coupled cytometric bead arrays, or similar assays.

[0108] By way of example, the screening method optionally comprises competitive binding between the one or more antibodies or modified antibodies provided herein and Donor specific antibodies (DSAs) in the biological sample with HLA molecules or epitopes thereof, which are optionally bound to a solid support. Optionally, the HLA molecules comprises one or more HLA alleles, wherein each allele is detectable (e.g., bound to a specific bead having a distinguishable label). The competitive binding assay requires discrimination between the one or more antibodies or modified antibodies described herein and the DSAs in the biological sample. Thus, the one or more antibodies or modified antibodies can be modified, for example, to have a non-human Fc region when the test antibodies (i.e., DSAs in the biological sample) have a human Fc region, and the test antibodies that bind one or more HLA alleles can be detected using a detectable secondary antibody specific for the human Fc. In this example, beads comprising bound one or more HLA alleles are incubated with the one or more antibodies or modified antibodies described herein (e.g., one or more of antibodies E07, L02, M07, 122, or any antibody provided in Table 2, or modified versions thereof, including any combination of the antibodies or modified antibodies provided herein). The beads are then washed to remove unbound one or more antibodies or modified antibodies. The beads are then incubated with the test sample to allow DSAs to bind to the beads and washed to remove unbound DSAs. DSAs bound to the beads are then detected using labeled secondary antibodies specific for the test antibodies (DSAs in the test sample).

[0109] In certain embodiments, the one or more antibodies or modified antibodies comprise a detectable label. The labeled one or more antibodies or modified antibodies are then distinguished from the test antibodies (DSAs) in the biological sample. The test antibodies can be labeled as described above using a secondary antibody that binds the test antibodies but not the one or more antibodies or modified antibodies as described herein.

[0110] Optionally, the method of reducing an immune response in a transplant recipient to a donor organ further comprises administering to the subject an immunosuppressive agent. The immunosuppressive agent may be, for example, tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, or steroids (such as glucocorticoids). The immunosuppressive agent may be administered to the subject before transplantation, including near or at the time of transplantation. For example, the immunosuppressive agent can be administered substantially simultaneously or nearly simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute, or less, before transplantation) or about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, or more, before transplantation. With simultaneous administration of the antibody or modified antibody, the immunosuppressant can be administered in the same composition or in a separate composition and by the same or different routes of administration (e.g., immunosuppressants may be administered orally whereas the antibody is administered by injection). The immunosuppressive agent can be administered to the subject at any time after transplantation. Optionally, the immunosuppressive agent can be administered to the subject upon clinical signs of a donor specific response determined by one skilled in the art.

[0111] Also provided herein is a method of treating a donor organ or tissue prior to or at the time of transplantation to reduce binding of donor specific antibodies in a transplant recipient comprising contacting the donor organ with the one or more antibodies or modified antibodies provided herein. The treating step is performed to block binding of antigens on the organ or tissue with B cells or DSAs produced by antibody secreting cells. The contacting step may be at any time, for example 1 to 5 minutes or up to 1 hour prior to transplantation or simultaneously with transplantation. The treating step may occur locally, wherein the donor organ or tissue is treated with the one or more antibodies or modified antibodies in isolation, or systemically, wherein the transplant donor is administered the one or more antibodies or modified antibodies prior to surgical removal of the organ or tissue from the donor.

[0112] Also provided herein is a method of screening a transplant recipient before or after transplantation to identify donor specific antibodies produced by the subject comprising obtaining a blood sample from the transplant recipient; contacting the blood sample with one or more antibodies or modified antibodies described herein; and determining whether the one or more antibodies or modified antibodies block binding of at least 50% of the subject’s donor specific antibodies to the subject’s human leukocyte antigen A*01 or A*01 :01. The methods can further include contacting the antibodies or modified antibodies with human leukocyte antigens A*01 or A*01 :01 prior to the contacting of the blood sample followed by determining whether the one or more antibodies block binding of the subject’s donor specific antibodies to the human leukocyte antigen A*01 or A*01:01. Optionally, the one or more antibodies or modified antibodies block binding of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of donor specific antibodies to the subject’s human leukocyte antigen A*01 or A*01:01.

[0113] As used herein, a blood sample refers to whole blood, serum, or plasma. The determining step can be selected from methods routine in the art. Methods can be used to determine whether the one or more antibodies or modified antibodies provided herein block binding of DSAs in a competitive binding assay. Optionally the assay comprises an ELISA, RIA, immunohistochemical, flow cytometry, FACS, IHC, FISH, proteonomic arrays, protein coupled cytometric bead arrays, or similar assays.

[0114] By way of example, the screening method optionally comprises competitive binding between the one or more antibodies or modified antibodies provided herein and donor specific antibodies (DSAs) in the biological sample with HLA molecules or epitopes thereof, which are optionally bound to a solid support. Optionally, the HLA molecules comprises one or more HLA alleles, wherein each allele is detectable (e.g., bound to a specific bead having a distinguishable label). The competitive binding assay requires discrimination between the one or more antibodies or modified antibodies described herein and the DSAs in the biological sample. Thus, the one or more antibodies or modified antibodies can be modified, for example, to have a non-human Fc region when the test antibodies (i.e., DSAs in the biological sample) have a human Fc region, and the test antibodies that bind one or more HLA alleles can be detected using a detectable secondary antibody specific for the human Fc. In this example, beads comprising bound one or more HLA alleles are incubated with the one or more antibodies or modified antibodies described herein (e.g., one or more of antibodies E07, L02, M07, 122, or any antibody provided in Table 2, or modified versions thereof, including any combination of the antibodies or modified antibodies provided herein). The beads may be washed to remove unbound one or more antibodies or modified antibodies. The beads are then incubated with the test sample to allow DSAs from the test sample to bind to the beads and washed to remove unbound DSAs. DSAs bound to the beads are then detected using labeled secondary antibodies specific for the test antibodies (DSAs in the test sample).

[0115] In certain embodiments, the one or more antibodies or modified antibodies comprise a detectable label. The labeled one or more antibodies or modified antibodies are then distinguished from the test antibodies (DSAs) in the biological sample. The test antibodies can be labeled as described above using a secondary antibody that binds the test antibodies but not the one or more antibodies or modified antibodies as described herein.

[0116] Optionally, the method of screening a transplant recipient further comprises treating the subject with the one or more antibodies or modified antibodies described herein that block at least 50%, optionally at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the subject’s donor specific antibodies from binding the subjects HLA A*01 or HLA A*01:01.

[0117] The herein provided kits can be used to carry out the methods of screening as described herein.

[0118] As used herein, treating refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a lessening in the severity or progression of an immune response, such as antibody-mediated rejection. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying or preventing an immune response, such as antibody mediated rejection. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0119] ADDITIONAL DEFINITIONS

[0120] As used in the specification and the appended claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an antibody includes two or more antibodies, and the like.

[0121] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0122] Ranges may be expressed herein as from about one particular value, and / or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0123] Optional or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which they do not.

[0124] EXAMPLES

[0125] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0126] Methods

[0127] Multi-color flow cytometry and sorting. The allograft nephrectomy specimen was mechanically and enzymatically digested to yield a single cell suspension. Mononuclear cells were isolated from peripheral blood using density-gradient centrifugation. The cell suspensions were stained with the following anti-human antibody reagents: CD19::V500 (BD, clone HIB19), CD71::BV605 (BD, clone M-A712), CD38::BV786 (BD, clone HIT2), IgD::FITC (BD), CDl lc::PE (Biolegend, clone Bul5), CD27::PE-CF594 (BD, clone M- T271), 7-AAD (BD), CD3::PE-Cy5 (Biolegend, clone HIT3a), CD14::PE-Cy5 (Invitrogen, clone 61D3), CD16::PE-Cy5 (BD, clone 3G8), CD56::PE-Cy5 (BD, clone B159). Cells were also stained with fluorescent-labeled tetramers of recombinant HLA-A* 01:01 (NIH tetramer core, loaded with peptide VTEHDTLLY (SEQ ID NO:416), in BV421 and AF647). Cells were sorted on a FACSAria (BD Biosciences) in the UAB Comprehensive Flow Cytometry Core as single cells for recombinant monoclonal expression and in bulk populations for next generation IgVH sequencing. Data were analyzed in FlowJo vl0.8.1 (BD).

[0128] Next generation sequencing of the IgVH repertoire (IgVH-Seq). IgVH sequencing was performed as described in Nellore et aL, Immunity, 56(4):847-863 (2023). After sorting B cells subsets directly into lysis buffer (Norgen Single Cell RNA Purification Kit) with 1% mercaptoethanol, lysates were snap frozen on dry ice and stored at -80°C. Total cellular RNA was extracted from bulk sorted B cell and ASC subsets according to the manufacturer’s protocol. cDNA preparation was performed (BioRad iScript) per manufacturer’s instructions, and DNA amplicon products were generated using Ig-specific variable and constant gene primers as described in Nellore et al., Immunity, 56(4):847-863 (2023). Samples were indexed (Nextera Index Kit, Illumina), purified, quantitated (KAPPA Library Quantitation Kit, Roche) and pooled into libraries. Following generation, libraries were denatured per manufacturer instructions (Illumina) and loaded onto a 600-cycle V3 MiSEQ cartridge (Illumina) for sequencing in the UAB Heflin Sequencing Core.

[0129] Phylogenetic analyses of IgVH-Seq data. IgVH sequencing data were processed and analyzed as described in Tipton, et al., Nat. Immunol. 16:755-765 (2015). Joined paired-end reads were assembled and quality filtered using FastQC scores. Using IMGT / HighV-QUEST, sequences were annotated, and productive sequences were utilized for downstream analyses. Sequences were clustered into lineages based upon IGHV and IGHJ gene identity, identical HCDR3 length, and HCDR3 nucleotide identity >85%. Downstream analyses were performed in Matlab (R2020a, The Mathworks Inc.) or in R (R core team). Phylogenetic trees were constructed using Phylip’s DNA parsimony (dnapars) tool (v3.695; adjusting settings 1, 4, 5, 6 and O setting the germline sequence as the outgroup (Phylip, v3.5 c). Phylogenetic trees were visualized using Cytoscape v3.8.2. (See, for example, Shannon et al., Genome Res. 13:2498-2504 (2003)).

[0130] Circos visualization of IgVn-Seq data. The outer numbering of Circos plots shows sequence counts for a given tissue-phenotype cell subset. The outer ring is colored (non-grey) for lineages contained in the top 20% of size-ranked lineages in a given tissue-phenotype cell subset. Links connect lineages shared between tissue-phenotype subsets; links are colored (non-grey) if the lineage is contained in the top 20% of size-ranked lineages in at least one tissue-phenotype cell subset. Alluvial ribbons depict connectivity of shared lineages, with the individual lineages ranked by size in each subset along the y-axis. Shared lineages are represented as ribbons with individual lineages ranked by size in each subset. The numbers of sorted cells for IgVH sequencing, numbers of unique non-singleton lineages and %D50 are indicated. %D50 = (# lineages in top 50% of subset A / # of lineages in the top 50% of subset B) x 100.

[0131] Single cell rmAb cloning, sequencing, and expression. Recombinant monoclonal antibodies (rmAbs) were generated as described in Nellore et al., Immunity, 56(4):847-863 (2023). Briefly, as shown in FIG. 1, cDNA was generated from mRNA isolated from single non-na’ive (CD19+IgDneg) dual HLA-A*01:01 tetramer-binding B cells that were index- sorted into hypotonic lysis buffer in 384- we 11 plates and stored at -80°C. PCR IgVH and IgVK / L amplicons, generated from the single cell cDNA, were cloned into expression vectors containing the constant regions of human IgGl, IgK, or IgL. Plasmids were sequenced by Sanger sequencing at the Genetic Resources Core Facility (RRID SCR 018669, Johns Hopkins University Department of Genetic Medicine, Baltimore, MD). IgH and IgL plasmids were co-transfected using polyethyleneimine (Polysciences) into 293FreeStyle cells (Invitrogen). Conditioned supernatants were screened for anti-HLA reactivity using HLA cytometric bead arrays described below. rmAbs were purified using Sepharose G Fast Flow (Cytiva) and rmAb concentrations were determined by UV spectrophotometry (Nanodrop, Thermo Fisher). For structural analysis by x-ray crystallography and cryo-electron microscopy, the heavy chain expression vector was modified using site-directed mutagenesis to remove the IgCn gene segment of each rmAb (JTK191B E07, JTK191B L02, JTK191B M07 and JTK191B I22). The resulting constructs were transfected into 293Freestyle cells as described above to produce antigen-binding fragments (Fabs). Fabs were purified using CaptureSelect IgG-CHl Affinity Matrix (Thermo Scientific).

[0132] Generation of modified and unmutated common ancestor (UCA) rmAbs. UCAs for each rmAb were generated by reverting all nucleotides encoding variable gene segments (VH, DH, JH, VK / L, JK / L) to the IMGT reference sequence of the gene segment alleles. Nontemplated nucleotides (N-additions) were not modified. Recombinant human IgGl mAb constructs were synthesized (Sino Biological) using the predicted UCA nucleotide sequences.

[0133] Immunogenetic analysis of rmAbs. IgVHand IgVK / L sequences from the cloned and verified A*01:01-specific rmAbs were submitted to IMGT / HighV-Quest for annotation. rmAbs were grouped into clusters based on identical VH, DH, JH, VK / L, JK / L gene assignments and identical HCDR3 and LCDR3 lengths. All 50 rmAb IgVH sequences were then directly compared to the sequences in the bulk-sorted kidney and blood B cell / ASC IgVH-Seq database. Any rmAb meeting the lineage criteria (same VH, DH, JH gene assignments, identical HCDR3 length and >85% NT identity across the HCDR) were assigned as members of the bulk B / ASC clonal lineages and these lineages were defined as A*01:01 specific. This approach resulted in the identification of 14 unique A* 01:01 -specific lineages, which each contained one or more A*01:01-specific rmAbs.

[0134] Plasma immunoglobulin proteomics. Recombinant HLA-A*01:01 was conjugated to CNBr-activated Sepharose (Cytiva) following manufacturer’s protocol. Recipient N006 plasma was diluted 1 / 10 in PBS, filtered using a 22mm syringe filter (Sigma-Aldrich) and passed over the HLA-A*01:01 column. Bound protein was eluted with 0.1M glycine pH 2.5, buffer-exchanged into PBS, concentrated to Img / mL, and stored at 4°C. The resulting affinity- purified A* 01 :01 -binding polyclonal plasma Abs (appAbs) were reduced with DTT, denatured at 70°C for 10 minutes (min) and separated on a 10% Bis-Tris Protein gel. IgH and IgL bands were visualized with colloidal Coomassie, excised from the gel, equilibrated in 100 mM ammonium bicarbonate (AmBc), and digested overnight with Trypsin Gold, Mass Spectrometry Grade (Promega) or endoproteinase AspN (New England BioLabs) following manufacturer’s instructions. Peptide digests were reconstituted in 0.1% Formic Acid / ddH2O at 0.1 pg / pL, injected (8 pL each) onto a 1260 Infinity nHPLC stack (Agilent Technologies), and separated using a 75 pm I.D. x 15 cm pulled tip C-18 column (Jupiter C-18 300 A, 5 pm, Phenomenex). This system runs in-line with a Thermo Q Exactive HFx mass spectrometer, equipped with a Nanospray FlexTM ion source (Thermo Fisher Scientific). Data were collected by the UAB Mass Spectrometry / Proteomics Core in CID mode. The nHPLC was configured with binary mobile phases that includes solvent A (0.1% FA in ddH2O), and solvent B (0.1% FA in 15% ddH2O / 85% ACN), programmed as follows: 10 min at 5% solvent B (2 pL / min, load), 90 min at 5%-40% solvent B (linear: 0.5 nL / min, analyze), 5 min at 70% solvent B (2 pL / min, wash), 10 min at 0% solvent B (2 pL / min, equilibrate). Following each parent ion scan (300-1200m / z at 60k resolution), fragmentation data (MS2) was collected on the top-most intense 10 ions at 7.5k resolution. For data dependent scans, charge state screening and dynamic exclusion was enabled with a repeat count of 2, repeat duration of 30 seconds (s), and exclusion duration of 90 s. Following LC-MS / MS, the data were processed, searched, filtered, grouped, and quantified, as described in Ludwig et al., Proteomics, 16:516-531 (2016). LC- MS / MS-derived peptide AA sequences were used to probe an AA sequence database that included all IgVHsequences derived from the kidney, blood and rmAb IgVH-seq libraries. Exact peptide matches to IgVHsequences were determined and members of the lineages identified were ranked by % of total AA matched. For visualization, peptide sequences were plotted by position within the IgVHsequence. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD043144 and 10.6019 / PXD043144..

[0135] HLA expression plasmids. Expression plasmids for HLA-A*01 :01 a-chain (pTCF5, Plasmid #180448) and b2m (pTCF158, Plasmid #180457) were purchased from Addgene. Single point mutations (V158A, R163T, or D166E) were introduced to this plasmid by site- directed PCR mutagenesis using complementary primers (Integrated DNA Technologies, Inc.) and Q5 High-Fidelity PCR Kit (New England Biolabs, E0555S). Plasmid DNA was sequence- verified for each HLA a-chain mutant.

[0136] Recombinant HLA production. Wildtype (WT) HLA-A*01 :01 or single residue mutants of HLA-A*01 :01 (V158A, R163T, D166E) were produced in-house as described in Altman and Davis, Curr. Protoc. Immunol., Chapter 17, Unit 17 (2003); Chatzileontiadou et al., STAR Protoc., 2(3):100635 (2021); and Garboczi etal., Proc. Natl. Acad. Sci., 89:3429-3433 (1992). BL21 (DE3) E. Coli chemically competent cells were transfected with expression plasmids for human 2m, WT A*01 :01 a-chain, or mutant A*01 :01 a-chain. Expressed recombinant proteins were extracted from inclusion bodies and refolded by dilution of the a-chain and 02m into refolding buffer containing excess peptide (VTEHDTLLY (SEQ ID NO:416), Biosynth Gardner, MA).

[0137] The HLA WT and mutant complexes were purified by ion exchange chromatography (DEAE- C) and size-exclusion chromatography, buffer exchanged into PBS, concentrated, and stored at 4°C. For experiments requiring biotinylated HLA, the proteins were enzymatically biotinylated with BirA enzyme (Avidity), then purified by ion-exchange and size-exclusion chromatography.

[0138] Generation ofHLA-coated microbeads. Custom cytometric bead arrays were generated as described in Nellore et al., Immunity, 56(4):847-863 (2023), using 4 pm and 5 pm carboxy functionalized array kits (Spherotech, PAK-4067-8K and PAK-5067-10K). Streptavidin (SA, Southern Biotech) was buffer-exchanged using PD-10 columns (Cytiva) into PBS and diluted to 2 mg / mL. To conjugate SA to the beads, lxlO8Spherotech beads were resuspended in 0.5 mL SA and 0.5 mL of 6 mM l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 0.05 M MES buffer pH 5.0 (Pierce). The reaction mixture was rotated at room temperature overnight and then quenched with 0.1 mL of 1 M tris pH 8.0. The beads were washed twice in 1 mL PBS and resuspended in PBS with 1% BSA and 0.005% NaNs and stored at 4°C. Recombinant, biotinylated HLA (see above) was incubated with SA conjugated beads at 1 mg / mL / IxlO8microbeads overnight at 4°C. Following HLA conjugation, the beads were washed twice in 1 mL PBS and resuspended in PBS with 1% BSA and 0.005% NaNs and stored at 4°C until used. Standard SA-HLA coated beads were used in all assays except Ab inhibition studies (see below), which used monoSA-HLA coated beads to minimize non-specific inhibition due to HLA packing density.

[0139] HLA cytometric bead arrays. Commercial (OneLambda, ThermoFisher or custom (see above)) HLA-coated microbeads were incubated for 15 minutes (min) with analytes (single IgGl rmAbs, patient plasma / serum, or appAbs) diluted in PBS or PBS with 1% BSA. Following incubation and washing, beads were incubated with anti-IgG::PE or anti- IgG::AF488 (2.5 pg / mL, Southern Biotech) in PBS with 1% BSA for 15 min. Beads were washed, resuspended in PBS and analyzed on a Cytoflex flow cytometer (Beckman Coulter Life Sciences). Data were analyzed in FlowJo vl 0.8.1 (BD). The geometric mean fluorescence intensities (gMFI) of binding by each rmAb to the different HLA allele-specific microbeads were calculated. Anti-influenza human IgGl rmAbs were used as negative controls. (See for example, Nellore el al., Immunity, 56(4):847-863 (2023)). The mean gMFI of the negative controls was subtracted from each allele-specific gMFI to obtain gMFI minus background (net gMFI). To select the minimum threshold for specific binding to HLA beads, the net gMFI of rmAb binding to three representative HLA alleles (A*01:01, A* 02:01, and A* 24: 02) was compared to the L-SPR-calculated KDof binding by the rmAb to each HLA allele. As 100% of tested rmAbs with an allele-specific net gMFI ≥40,000 had measurable allele-specific binding (KD< 4.3 pM) by L-SPR, specific binding to the HLA beads was defined as a net gMFI ≥40,000.

[0140] Binding of rmAbs to HLA A *01:01 mutants. MonoSA-conjugated microbeads that were loaded with WT A* 01:01 or A*01 :01 mutants were generated (see above), stained with analytes (rmAbs, appAbs, or sera / plasma) at multiple concentrations, and the gMFI of analyte binding to the beads displaying WT or mutant A*01:01 was determined. The half maximal effective concentration (EC50) of rmAb binding to each A*01 :01 mutant was calculated using the sigmoidal, 4PL, x=concentration model in GraphPad Prism, with constraints (bottom = 0, top = 100). The gMFI of rmAb to A*01:01 mutant-coated beads was normalized to the maximum gMFI according to the following formula: % maximum gMFI = (gMFI of sample / highest gMFI for given protein) x 100%. To ensure accurate curve fitting, prozone points were omitted from the analysis. EC50 values were calculated from at least two technical replicates for each experiment. Serum anti-HLA Ab activity was interpolated from a standard curve using the pan-HLA Class I-reactive Ab W6 / 32 (Biolegend, 15.6 ng / mL to 2000 ng / mL) binding to WT A*01 :01 and mutant HLA A*01:01 beads and corrected for dilution factor. The resulting standard curves were each fitted to a sigmoidal, 4PL, x=concentration curve. Each serum sample was initially assayed at a 1 :500 dilution. In cases where the interpolated value for a given sample was outside of the vertical range of the standard curve, the serum sample was retested at a higher (1 :4,000 or 1 : 12,000) dilution.

[0141] Chimeric rmAb inhibition assays. Mouse IgGl Fc chimeric (c) Abs (cE07 and cL02) were generated by fusing the human IgVn domain of E07 or L02 to the mouse IgGl constant region. The human IgVi domain of E07 or L02 was then fused to either the mouse constant region kappa (for L02) or lambda (for E07) (Sino Biological) and the chimeric IgH and IgL proteins were co-expressed in 293 Freestyle cells. For Ab competitive inhibition analyses, monoSA-conjugated beads loaded with A*01 :01 were produced (see above) then diluted in PBS alone (non-blocked beads) or diluted in PBS containing 5 pg / mL recombinant mouse chimeric cE07 or cL02 Abs (blocked beads) and then incubated for 10 min at RT. Analytes, including test human rmAbs, polyclonal appAbs, or polyclonal serum / plasma, were added at varying concentrations to the blocked and unblocked beads and bead suspensions were incubated for an additional 10 min. The beads were washed in PBS, stained for 10 min with anti-human IgG FC:PE (multi-species absorbed, Southern Biotech) diluted in PBS with 1% BSA, then washed and analyzed as described above. Percent inhibition of binding to the beads was calculated as follows: (unblocked gMFI - blocked gMFI) / (unblocked gMFI) x 100. All negative percent (%) values in analyses were set to 0. Percent inhibition values were calculated from at least two technical replicates for all experiments.

[0142] Clinical anti-HLA Antibody testing. Clinical anti-HLA Ab testing for recipient N006 and the cohort of n=l l solid organ transplant recipients was performed by the UAB Histocompatibility Laboratory using its standard clinical assay. For the clinical MFI data, IgG Abs from patient sera were collected using a Melon IgG spin column and then analyzed neat using the OneLambda HLA arrays, according to manufacturer instructions (OneLambda, ThermoFisher). Proprietary software was used for the MFI calculation with a MFI ≥5,000 used as the clinical threshold for a positive reading. HLA-specific Ab data was obtained from the patients’ electronic medical record. Clinical HLA typing by next-generation sequencing or by imputation. HLA typing of both donor (donor to recipient N006) and recipient (subject N006) was performed by the UAB Histocompatibility Laboratory. Genomic DNA was extracted from peripheral blood samples using the Qiagen EZ1 DNA blood kit via the Buffy coat protocol. Library construction and enrichment were performed per manufacturer’s protocols for the CareDx AlloSeq Tx 17 assay and libraries were run on the Illumina MiSeq. Propietary CareDx software, AlloSeq Assign, vl.0.3 (CareDX, Stockholm, Sweden) utilized references from the Immuno Polymorphism Database-ImMunoGeneTics project / HLA Database (IPD-IMGT / HLA database) v3.47.0.0 to make allele assignments for all HLA Class I and Class II loci. For donors and recipients in the n=l 1 cohort that were not HLA typed by next-generation sequencing, 4 digit HLA-A alleles for recipient and / or donor were imputed based upon ethnicity and population frequencies as described in Gragert et al., Human Immunology, 74:1313-1320 (2013).

[0143] High-throughput localized surface plasmon resonance. Localized surface plasmon resonance (L-SPR) data were collected using a 1 : 1 referencing system on the Nicoya Alto HT-SPR instrument according to manufacturer’s recommendations and as described in Woodruff et al., Nature, 611:139-147 (2022). Briefly, biotinylated recombinant HLA- A*01 :01 (500 pg / mL) or HLA-A*24:02 (200 pg / mL) was resuspended in PBS containing 0.05% Tween-20 and immobilized on an EDC / NHS-activated carboxyl sensor (Nicoya) for 20 min. The rmAbs (analytes) were diluted to 1.3 pM in PBS containing 0.05% Tween-20. Single-cycle kinetic analyses were performed at 25°C with five threefold analyte dilutions, with maximum concentrations of 433 nM. Kinetic fitting was performed using a 1:1 Langmuir model within the Nicoya user portal. For datasets that poorly fit with a 1 : 1 Langmuir model, an affinity fit model was used (TraceDrawer, Ridgeview Instruments). The limit of detection for the equilibrium constant (KD) was estimated to be 4.3 pM, or 10 times the highest concentration of analyte tested (433 nM). Recombinant mAbs with responses below 50 plasmon resonance response units or without monotonic increases across increasing concentrations were judged to not have measurable binding by L-SPR. For visualization and for calculations of fold-change in affinity, if a rmAb had no detectable binding by L-SPR (KD >4.3 pM), a KD of 5 pM was used.

[0144] X-ray crystallography and CryoEM sample preparation. HLA / Fab complexes were generated by mixing recombinant, unbiotinylated HLA-A*01 :01 (described above) with either one Fab (1:1 molar ratio for E07 / A*01:01 or I22 / A*01:01, on ice) or with two Fabs sequentially (1:1 :1 for L02 / M07 / A*01 :01). Complexes were purified using size-exclusion chromatography on a Superdex S200 column in PBS. Complexes were concentrated and prepared for subsequent structural studies.

[0145] X-ray crystallization, data collection, structure determination, and refinement. Purified E07 / A*01:01 complexes were concentrated to 9.8 mg / mL and screened for crystallization conditions against commercially available kits using a Mosquito liquid handling robot (SPT Labtech). Crystallization conditions were optimized, and diffraction quality crystals were grown in 1.015 M sodium citrate tribasic dihydrate, 0.1 M sodium cacodylate pH 6.5 at a 1 :1 ratio of protein to precipitating agent. Crystals were cryoprotected in crystallization solution supplemented with 20% (v / v) ethylene glycol and frozen. Diffraction data was collected at SER-CAT beamline 22-ID and processed with HKL2000. Data analysis was performed at UAB. Crystals exhibited a pseudo-cubic symmetry but had true C2 symmetry. Initial phases were solved by molecular replacement with PHASER using prior models of HL A (PDB ID: 6AT9 (see, for example, Toor et al., Front Immunol. 9: 99 (2018)), antibody domains:VH (PDB ID: 6PZH (se,e for example, Zhu et al., Cell Host Microbe, 26: 729-738 (2019)), VL (PDB ID: 4HK0 (see for example, Schmidt et al., Proc. Acad. Natl. Sci., 110:264-269 (2013)), CHI (PDB ID: 6AZZ (see, for example, Scally et al., Nat. Commun., 8:1568 (2017)). After identifying a trimeric assembly of HLA-Fab complexes, the assembly was used to find three additional copies of the assembly, yielding an asymmetric unit that contained 12 copies of HLA and 12 copies of the Fab. Models were iteratively rebuilt with COOT. (See for example, Emsley et al., Acta. Crystallogr. D. Bio. Crystr ollogr., 66:486-501 (2010) and refined with phenix.refine. (See for example, Adams et al., Acta. Crystallogr. D. Bio. Crystrollogr., 66:213-221 (2010). Though 12 copies of each protein could be identified in the asymmetric unit, domain a3 for some HLA and 2m had degenerate density following refinement due to high B-factors, likely a result of crystal packing, and were thus removed from the final model. After removal, electron density from Fo-Fc difference maps showed density for the additional domains, confirming their presence in the lattice. The final model contains six copies of the complete HLA-Fab complex, six additional copies of the Fab, three copies of the HLA domain with 3 removed, and two copies of 02m. Structure factors and final refined coordinates were deposited in the PDB (8T7R). X- ray data collection and refinement statistics are provided in Table 3A.

[0146] Table 3A. X-ray data collection and refinement statistics

[0147] Table 3B. CryoEM data collection and refinement parameters.

[0148] CryoEM grid preparation and data collection. The L02 / M07 / A*01:01 complex was concentrated to 3.04 mg / mL in PBS. Grids for electron cryo-microscopy were prepared by placing 3mL of solution on a 2 / 1-3T C-flat grid (R2 / 1 thick carbon, 300 mesh, Protochips), which was plunged into liquid ethane with a Mark IV Vitrobot. The grids were clipped and transferred to a G3 FEI-Krios electron microscope (La Jolla Institute for Immunology), which is equipped with a Gatan Bio-quantum energy filter with a 20eV slit. Images were detected on a K3 camera in counting mode (not super-resolution). The electron beam intensity was ~15e- / pixel*s and 50 fractions were collected for every image, giving the specimen le / A*A*frame. The beam size was 900nm, which allowed for collection of 6 images per hole in the sample.

[0149] CryoEM data processing, model building and refinement. CryoEM data processing was performed by the UAB CryoEM Facility. The images were pre-processed with Warp. (See for example, Tegunov and Cramer, Nat. Methods, 16L1146-1152 (2019)) where the images were corrected for movement, CTF parameters were estimated, and an initial set of particles was picked. CryoSPARC v4 was used for data processing. (See, for example, Punjani et al., Nat. Methods, 14:290-296 (2017)). Pre-processing was repeated using patch motion correction and CTF estimation. From 14,572 exposures, blob picking and 2D classification yielded 93,263 preliminary particles for Topaz training (FIG. 2A).Topaz autopicked 804,662 particles, which were initially downsampled to 2.64A / pix and cleaned via 2D classification. The remaining particles were classified into four classes using ab-initio 3D reconstruction. This was done in quadruplicate for three iterations, keeping any particles that contributed to a good class. Finally, 252,746 particles from 3D classes corresponding to the L02 / M07 / A*01:01 complex were kept. After reverting to the original 0.66 A / pix particles, these were refined using non-uniform refinement to a resolution of 3.14A (FIG. 2B-C). To initiate atomic model building, AA sequences were input to ColabFold via ChimeraX to generate AlphaFold predicted models. (See for example, Mardita et al., Nat. Methods, 17:1214-1221 (2020); Pettersen et al., Protein Sci., 30:70-82 (2021); Jumper et al., Nature, 596:583-589 (2021)). M07 and L02 were differentiated based on AAs with bulky side chains present in either, but not both, which could be clearly identified in the reconstruction. AlphaFold initial models were rigid-body fitted to the reconstruction using ChimeraX and refined in real space using Coot (FIG. 2). (See for example, Emsley et al., Acta. Crystallogr. D. Bio. Crystallogr., 66:486-501 (2010)). Phenix was used for global real-space refinement and model validation. (See for example, Afonine et al., Acta. Crystallogr. D. Struct. Bio. Crystallogr., 74: 531-544 (2018)). See Table 3B for CryoEM data collection and refinement parameters. Similar cryoEM methods were used to determine structure of HLA-A*01 :01 in complex with 122 (FIG. 16) and L02.

[0150] Structural analyses. Properties of Fab / HLA binding interfaces were analyzed using UCSF ChimeraX. (See for example, Pettersen et al., Protein Sci., 30:70-82 (2021)). Interacting residues at the Fab:HLA interface were identified using a heavy-atom (nonhydrogen atom) distance cutoff of ≤5 Å. Epitopic residues were defined as those with an interatomic distance between a heavy atom in the Fab and a heavy atom in the HL A complex ≤5 Å or any residues in the HLA complex with solvent accessible surface area that was buried in the interface between the Fab and HLA- A*01:01. Hydrogen bonds were identified using UCSF ChimeraX hbond command with default criteria. (See for example, Pettersen et al., Protein Sci., 30:70-82 (2021)). In silico methods were used to observe interactions of Fab E07 with HLA complexes harboring mutations V158A, R163T, or D166E. Individual mutants were made with Coot. Complexes were subjected to the molecular dynamics energy minimization routine in YASARA as described in Krieger et al., Proteins, 77 Suppl 9:114- 122. (2009).

[0151] Immunofluorescent histology of allograft specimens. After obtaining the transplant nephrectomy specimen, tissue blocks were embedded in Tissue-Tek O.C.T. Compound (Sakura) and frozen in liquid nitrogen. Tissue sectioning (6 pm) was performed on a cryostat (Leica CM1860), and sections were stained with three different panels of anti-human secondary antibodies, including CD21 ::FITC (Biolegend, clone Bu32), CD19::APC (BD, clone HIB19), CD21::FITC (Biolegend, clone Bu32), CD4::AF488 (Biolegend, clone OKT4), CD19::PE (Biolegend, clone HIB19), Ki67::APC (Biolegend, clone Ki-67), anti- Ig::AF488 (Southern Biotech, polyclonal), CD38::APC (Biolegend, clone HIT2), and DAPI. Images were acquired on a Nikon Eclipse Ti microscope with the eyepiece at 1 Ox, mirror at lx, and objective of 20x (for total 200x) or 40x (for total 400x). Images were exported at 300dpi as *.TIFF files. Originals are saved as *.ND2 files including metadata and exposure times. For FIG. 3A-C, exposure times (in milliseconds) for each channel (DAPI, FITC, PE, APC) are listed: FIG. 3A (500, 3000, 6000, 10000), FIG. 3B (2000, 3000, 8000, 10000), FIG. 3C left (60, 70, 1000, 600), FIG. 3C right (250, 70, 1400, 800). The tonal range of RGB was reduced in Adobe Photoshop (Adobe Inc., San Jose, CA) to increase brightness and contrast, with settings uniformly applied across all images utilizing the same staining panel.

[0152] Analysis of HLA residue properties. For consistency across structural models, a previously reported structure of HLA A*01 :01 (PDB: 3BO8) (See for example, Kumar et aL, Protein Sci., 18:37-49 (2009)) was used for calculations of HLA A*01 :01 residue solvent accessibility in the unbound state by PISA and Ellipro scores for residue protrusion. Physicochemical mismatches were compared as described in Kirk et al., Am. J. Transplant, 7:1464-170 (2007) for hydrophilicity and electrostatic mismatch, and AA substitution similarity scores were used for dissimilarity calculation as described in Risler et al., J. Mol Biol., 204:1019-1029 (1988). To calculate residue distances from the center of the peptide- binding groove (PBG), the HLA A*01:01 (PDB: 3BO8) structure described above was used and the center of the PBG was defined as the a-carbon of the 5th residue (T5) in the peptide nonamer (EADPTGHSY (SEQ ID NO:417)) presented within the PBG. Distances were then calculated between the peptide T5 residue a-carbon and the a-carbon of each AA in A*01 :01 using UCSF ChimeraX. (See for example, Pettersen et al., Protein Sci., 30:70-82 (2021)).

[0153] B cell epitope prediction using computational tools. B cell epitopes within the HLA- A*01 :01 protein were predicted using GraphBepi as described in Zeng et al., Bioinformatics, 39:10.1093 / bioinformatics / btadl87 (2023) and DiscoTope-3.0 as described in Hoie et al., Front Immunology, 15:1322712. 10.3389 / fimmu.2024.1322712 (2024). For both tools, the previously reported (see Kumar et al., Protein Science, 18:37-49 (2009)) HLA-A*01:01 (PDB: 3BO8) structure was used. For GraphBepi, default settings were used. For DiscoTope- 3.0, the epitope confidence threshold was set to 1.50.

[0154] HLA genotyped cohort transplant simulation. Previously reported (see Moore et al., Human Immunology, 79:821-822 (2018)) HLA genotyping data derived from a cohort of 310 United States subjects and selected 247 subjects who did not express an A*01 :01 self-allele were examined. The AA residues that were mismatched between the self-HLA-A alleles expressed by each individual and the non-self A*01 :01 protein were identified and the total number of solvent-accessible residues that were classified as 2MM and located within the AH-PBG region was determined for each subject. The percentage of these 2MMs that were contained within the E07 and L02 epitopes was calculated. Residues were classified as solvent-accessible if the residue non-buried surface area was ≥50 Å2. Statistical analyses. All statistical comparisons were performed in Prism (GraphPad Software). Comparisons of an observed distribution with the expected distribution were performed using the binomial test and the confidence interval of the proportion was calculated using the hybrid Wilson / Brown method in Prism. Following confirmation of distribution normality by the Shapiro-Wilk test, comparisons of group means was made using an unpaired t-test. If distributions were assessed to be non-normal, group ranks were compared via the Mann- Whitney test. Spearman correlations were used to assess association between two nonparametric variables. Hierarchical clustering, heatmap plots, and other visualizations were created using R v4.2.0 (R Core Team 2022), Matlab (R2020a, The Mathworks Inc., Natick, MA), UCSF ChimeraX vl.5, Cytoscape v3.8.2, FlowJo vlO.8.1 (BD, Franklin Lakes, NJ), Prism v9.5.1 (GraphPad Software, LLC., Boston, MA), or Biorender.com (Toronto, Canada).

[0155] Data and Code Availability. IgVH sequencing data has been deposited on GEO (GSE235533). CryoEM data has been deposited in PDB, EMDB, and EMPIAR (8T6M, EMD-41072, EMPIAR-11591). X-ray crystallography data has been deposited in PDB (8T7R). Proteomics data is deposited in ProteomXchange (PXD043144). Original code for the plasma proteomics analysis is deposited on Zenodo (10.5281 / zenodo.8039145).

[0156] Results

[0157] Transplant recipient kidney and blood B lineage cells display features of antigen-driven selection.

[0158] To evaluate the contribution of alloreactive B cells to allograft rejection, the tissue and circulating donor HLA-specific B lineage response was examined in a kidney allograft recipient (subject N006) who presented with histopathologic allograft injury consistent with antibody mediated rejection (AMR) and expressed high levels of circulating donor specific antibodies (DS A) directed against the mismatched HLA allele (HLA-A*01:01) expressed on the donor kidney. Following surgical nephrectomy to remove the rejected kidney from recipient N006, immunohistologic and flow cytometric analyses were performed on tissue sections and cells isolated from the allograft. Tertiary lymphoid structures were not identified in this allograft (FIG. 3 A-C). However, clusters containing T and B cells (FIG. 3A), proliferating B cells (FIG. 3B), and antibody secreting cells (ASCs, FIG. 3C) were observed. Furthermore, multiple B cell subsets were identified, including IgDnegCD27+ memory B cells, IgDnegCD27neg (double negative, DN) B cells, and CD27hiCD38hi ASCs (FIG. 3D), in the graft and many of these intragraft B cells expressed the memory B cell (Bmem) activation marker CD71 (FIG. 3E). Thus, the allograft contained activated, proliferating antigen-experienced B cells and ASCs.

[0159] To address whether the B cells and ASCs found within the allograft were distinct from the systemic circulating pool of ASCs and B cells, bulk populations of ASCs and antigen-experienced Bmem and DN subsets from the blood and the kidney (FIG. 4A-B) were sort-purified and the B cell receptor (BCR) Ig heavy (IgH) chain variable domains (IgVH) of these cells were sequenced. The IgVHsequences were assigned to clonal lineages, which was defined as sharing IGHV and IGHJ gene segments, having identical IgH chain complementarity determining region 3 (HCDR3) lengths, and exhibiting >85% nucleotide identity across the HCDR3. Then, clonal relationships were analyzed between the B cells and ASCs found in circulation and those in the allograft. It was observed that extensive clonal expansion of B lineage cells within the kidney allograft, with rich connections between the ASC, Bmem, and DN B cell subsets (FIG. 3F). Moreover, and in contrast to the low frequency of shared clonal lineages between blood and tissues isolated from a nontransplanted individual (FIG. 4C), numerous shared lineages were identified between the B cells in the graft and blood of recipient N006 (FIG. 3G, FIG. 4C). It was further observed that most lineages of B cells and ASCs present in the kidney were also found in the blood (FIG. 3H). Next, the isotype and SHM profile of the BCRs expressed by the kidney and blood B cells was assessed. Most B cells in both sites were class-switched (FIG. 4D), and the B cells and ASCs from both sites had undergone extensive SHM (FIG. 4E). Thus, the B cells in the rejected kidney and blood of a transplant recipient undergoing AMR were isotype-switched, clonally expanded, shared, and mutated - all consistent with an ongoing robust adaptive immune response that spanned the allograft and blood.

[0160] Class-switched, mutated alloHLA-specific Bmem and ASCs accumulate in the allograft and blood.

[0161] Since standard clinical lab assays revealed that DSA for the allograft-derived HLA- A*01 :01 was readily detected in the blood of transplant recipient N006 (FIG. 31), it was hypothesized that some of the allograft-infiltrating, clonally expanded and affinity-matured B cells would be specific for the mismatched A*01:01 donor HLA allele. To test this hypothesis, fluorochrome-labeled tetramers of recombinant HLA-A*01:01 were used to sort- purify single A*01:01-specific B cells from the allograft and the peripheral blood (FIG. 3J, FIG. 5A-B) and then the BCR IgH and Ig light (IgL) chain genes were cloned, sequenced, and expressed as recombinant human IgGl monoclonal Abs (rmAbs, schematic in FIG. 1). Using A*01:01-coated microbeads, it was confirmed that 50 rmAbs, which were cloned from sorted A*01:01-specific Bmem (43 / 50) and DN B cells (7 / 50) isolated from the peripheral blood and kidney, bound to the mismatched A*01 :01 donor HLA protein (FIG. 3K). Next, the IgVHsequences from the 50 A*01 :01-specific rmAbs were used to evaluate whether the rmAbs derived from the A*01 :01-specific B cells were clonally related to any of the expanded B cell lineages identified in the bulk kidney and blood IgVH-Seq dataset. If one or more A*01 :01-specific rmAbs shared IGHV and IGHJ gene segments, identical HCDR3 lengths, and >85% nucleotide identity across the HCDR3 with a clonal lineage identified in the bulk IgVH-Seq dataset, the rmAbs were assigned to this clonal lineage and the entire lineage was classified as A*01:01-specific. Using this approach, 14 distinct A*01:01-specific lineages were identified that linked the 50 A*01:01-specific rmAbs and the clonally related BCR IgVHsequences (n=4142 sequences) derived from the bulk kidney and blood Bmem, DN B cells and ASCs. It was observed that the A*01 :01 -specific clonal lineages had undergone extensive SHM (FIG. 3L) and that Bmem, ASCs and DN cells in the A*01:01- specific lineages were each heavily mutated (FIG. 5C-D). While the total blood IgVH-Seq repertoire included many IgA sequences (FIG. 4D), this was not observed in the A*01 flspecific lineages, which showed almost exclusive IgG utilization across all subsets in both blood and kidney (FIG. 3M). Thus, multiple lineages of IgG-switched and highly somatically mutated B cells, specific for the mismatched donor A*01 :01, were present in the blood and rejected kidney.

[0162] Clonal lineages of allospecific affinity-matured B cells are shared across kidney and blood.

[0163] The somatic hypermutation profile of the A*01:01-specific B cell lineages was used to evaluate the evolution of the alloresponse to donor HLA. Many examples of lineages that contained multiple B lineage subsets (FIG. 6A-C) that were found in both kidney and blood were identified. As expected, these B cells and ASCs exhibited increasing numbers of mutations within the clonal lineage tree (FIG. 6A-B). Moreover, 8 of the 14 A*01:01-specific lineages were each individually responsible for at least 2.5% of IgVHsequences in the entire A*01 :01-specific IgVH-Seq repertoire (FIG. 6D) and together, these 8 clonal lineages collectively accounted for 93.0% of all A*01 :01-specific sequences in the IgVH-seq database. At least two different types of B lineage populations were found in 10 / 14 lineages (FIG. 6E), with the most represented subsets including kidney ASC (present in 11 lineages) and kidney Bmem (present in 8 lineages, FIG. 6E). Next, localized surface plasmon resonance was used (L-SPR, FIG. 6F) to measure the binding affinities ( KD) of the 50 rmAbs specific for A*01 :01. It was determined that the median KDof binding to A*01 :01 by the 50 rmAbs derived from Bmem and DN B cells was 2.1 nM (FIG. 6G). To confirm that this high-affinity binding was due to the accumulation of mutations within the BCR and was not germline- encoded, rmAbs encoding the inferred unmutated common ancestors (UCAs) of 9 different A*01 :01-reactive lineages and assayed binding of the UCA rmAbs to A*01 :01 were synthesized. As shown in FIG. 6H, the KDS of two mutated rmAbs, which were members of either Lin4 (rmAb D01) or Lin12 (rmAb L02), were between 100- to 1000-fold lower (i.e. higher affinity) than the KDS of the rmAbs encoding the inferred UCA for Lin4 and Lin12. Similarly, significant (p<0.0001) increases in A*01:01 affinity (lower KDvalues) for all 36 rmAbs present in 9 different lineages when compared to the UCA rmAbs for each of these nine lineages were observed (FIG. 61). Interestingly, the UCAs from 5 / 9 lineages showed no detectable binding to A*01:01 by L-SPR (FIG. 6J). These data support the conclusion that the somatically mutated A*01:01 donor-specific B cells have undergone affinity maturation and have been selected for high-affinity clones.

[0164] Linking the allospecific tissue and circulating B cells to the systemic alloantibody response.

[0165] Next, the Ig proteome of the systemic plasma A*01:01-specific polyclonal Ab from recipient N006 was compared to the BCR repertoire of the A*01 :01-specific B cells and ASCs from kidney and blood of recipient N006. First affinity-purification of the polyclonal A*01 :01-specific Abs (appAbs) from plasma of recipient N006 was performed and then the appAbs were enzymatically digested to peptides that were subsequently analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The A*01 :01 -specific appAb peptide sequences were aligned to the AA sequences encoded by the IgVHnucleotide sequences derived from the A*01 :01-specific BCR clonal lineages identified in recipient N006. Using this approach, overlapping A*01 :01-specific appAb peptides were identified that matched the sequences of the BCRs expressed by the A*01:01-specific B cells and ASCs found in recipient N006 kidney and blood. Importantly, this overlap included matched identical sequences across the hypervariable HCDR3 regions of the rmAbs derived from recipient N006 A*01 :01-specific B cells. For example, AA sequences of peptides recovered from the A*01:01-specific appAbs were identical to IgVHAA sequences from two different Lin4 rmAbs (D01 and G22) cloned from blood Bmem (FIG. 7A-C). This finding was not limited to Lin4 as many circulating polyclonal appAb peptide sequences were identified that could be mapped to A*01:01 -specific rmAbs cloned from Lin 14 and Lin6 blood Bmem (FIG. 7D-E). In fact, appAb peptides were mapped to IgVHsequences from 4 different A*01 flspecific lineages (FIG. 7F) that included kidney- and blood-derived B cells and ASCs. Thus, circulating DSA was clonally related to the ongoing donor-specific blood and kidney B cell and ASC responses.

[0166] Dominant HLA reactivity patterns within the allospeciflc B cell lineages.

[0167] The data showed that the B cell and ASC response to the donor HLA-A alloantigen was robust and included at least 14 distinct B cell clonal lineages. These data suggested that the genetically diverse alloreactive B cells might be specific for different epitopes within the mismatched HLA protein. If so, it was predicted that the rmAbs derived from distinct clonal lineages would exhibit differing patterns of reactivity for other third party (neither host nor donor) HLA-A proteins that were also mismatched to self-HLA at one or more AA residues. Thirteen (13) residues were identified that were double mismatched (2MM) between the HLA-A expressed by the donor kidney (A*01:01) and the self-HLA expressed by recipient N006 (A*24:02 / A*30:01). Of the 13 2MM AA residues, 7 AA were solvent-accessible (defined as ≥50 Å2solvent-accessible surface area) and could potentially be targeted by an Ab (FIG. 8A). Therefore, it was hypothesized that the collection of 50 rmAbs would exhibit different patterns of HLA reactivity that targeted these seven individual mismatched residues. To test this, the binding of the 50 A*01 :01-specific rmAbs against a panel of 21 different HLA-A molecules was examined (FIG. 9A, FIG. 8B). Consistent with the hypothesis, it was observed that the rmAbs displayed 17 distinct HLA-A reactivity patterns (FIG. 9A, FIG. 8C). Importantly, when the reactivity profiles of the 50 rmAbs cloned from Bmem and DN isolated from recipient N006 was compared to the reactivity profile of the A*01 :01-specific polyclonal appAbs present in circulation of the same individual, it was found that the patterns were strikingly similar - with the rmAbs and the appAbs showing the highest reactivity towards three HLA-A proteins: A*01:01, A*36:01 and A*80:01 (FIG. 9B). Thus, the HLA reactivity profile of the systemic appAbs was very similar to the aggregated profile of the collection of rmAbs that were cloned from 50 A*01 :01-specific blood and kidney B cells.

[0168] Although multiple reactivity patterns among the A*01:01-specific rmAbs was observed (FIG. 9A- B, FIG. 8C), some patterns were more highly represented within the rmAb collection. For example, the A*01:01-monospecific reactivity pattern (referred to asA1+) included 17 / 50 (34.0%) rmAbs that bound A*01:01 alone (FIG. 9C). The 17 rmAbs exhibiting an A1+monospecific reactivity pattern were derived from eight distinct clonal lineages (FIG. 10A). Similarly, 5 / 50 (10.0%) rmAbs spread across two distinct clonal lineages (Lin4 and Lin12) exhibited an A*01:01+A*29:02+A*36:01+A*80:01+(A1+A29+A36+A80+) reactivity pattern (FIG. 9C, FIG. 10B). Finally, a third lineage (Linl), which contained ten clonally related rmAbs, exhibited consensus binding to six HLA-A alleles: A*01 :01+A*03:01+A*l l:01+A*24:02+A*36:01+A*80:01+(FIG. 9C, FIG. IOC).

[0169] Next, it was investigated whether specific HLA reactivity patterns were associated with the largest lineages in the A*01 :01-specific IgVH-seq database. Lin4, which represented 20.0% of all IgVHsequences in the A*01:01 specific IgVH-seq database and contained four rmAbs (FIG. 9D), displayed A1+A29+A36+A80+reactivity (FIG. 10B). Lin12, which also displayed A1+A29+A36+A80+reactivity (FIG. 10B), contained one rmAb and comprised 19.0% of A*01:01 -specific IgVHsequences (FIG. 9D). Thus, five A1+A29+A36+A80+rmAbs were linked to two clonal lineages that collectively constituted 39.0% (FIG. 9D) of all A*01 :01 -specific IgVHsequences. Similarly, the eight clonal lineages containing the 17 A1+monospecific rmAbs (FIG. 10A) represented 36.9% of the entire A*01:01-specific IgVHrepertoire (FIG. 9D). Thus, despite appreciable diversity in the HLA-reactivity patterns within the A*01:01 -specific B cell response, two patterns of HLA reactivity, which together encompassed 75.9% of all A*01:01-specific IgVHsequences in the database, dominated the A*01 :01-specific IgVH-Seq repertoire. These results suggested that the B cell and Ab response to the mismatched HLA-A protein might be focused on a limited number of immunodominant HLA-A epitopes.

[0170] HLA epitope recognition by immunodominant allospeciflc B cells.

[0171] The clonal lineage and HLA reactivity data suggested strongly that the B cell alloresponse might be focused on a relatively small number of highly “immunogenic” mismatched residues. To test this idea, X-ray crystallography and cryo-electron microscopy (cryoEM) were used to determine the structures of 4 representative rmAb / HLA-A*01 :01 complexes. These included the Lin2 E07 rmAb (FIG. 9E), which exhibited the immunodominant A1+monospecific pattern of reactivity (FIG. 10A) and was derived from a kidney A*01:01-specific Bmem, and the Lin 12 L02 rmAb (FIG. 9F), which exhibited the immunodominant A1+A29+A36+A80+pattern of reactivity (FIG. 10B) and was also derived from a kidney Bmem. The kidney Bmem-derived rmAb M07 (FIG. 9G), which was part of Linl that included 20.0% (10 / 50) of the rmAb collection and exhibited the A1+A3+A11+A24+A36+A80+reactivity profile was examined (FIG. 10C). Finally, rmAb 122 (FIG. 16), which was part of the large Lin6 lineage (FIG. 9D) was examined.

[0172] To characterize the structural epitope(s) in HLA-A*01:01, the epitope was defined as residues with an interatomic distance of ≤5 Å between a heavy atom in the Fab and a heavy atom in the HLA complex or any residues in the HLA complex with solvent-accessible surface area that was buried in whole or in part in the interface between the Fab and HLA- A* 01 :01. Using these criteria, it was found that the footprints of the HLA epitopes bound by the four rmAbs were consistent with the size of typical protein epitopes (as discussed in Ramaraj et al., Biochimica et Biophysica Acta 1824:520-532 (2012)) with the rmAbs burying 912 Å2(E07), 582 Å2(L02), 775 Å2(M07) and 683 Å2(122) of solvent accessible surface area within the HLA-A*01 :01 a-chain (FIG. 9E-G, FIG. 16A). The number of AA residues (n=14-22 A*01:01 a-chain residues) present in each of the HLA epitopes (FIG. 9E-G and FIG. 16A) was similar to other previously reported (see for example Ramaraj et al., Biochimica et Biophysica Acta 1824:520-532 (2012) and Akbar et al., Cell Reports 34:108856 (2021)) protein epitopes. Consistent with the different HLA reactivity patterns exhibited by each rmAb, it was found that the rmAbs bound four distinct epitopes on

[0173] A* 01 :01, with little overlap in the binding footprints (FIG. 9H and FIG 16A,B). For example, the HLA-A*01:01 epitopes recognized by rmAbs E07 (Lin2, A1+monoreactivity pattern) and M07 (Lin 1, A1+A3+A11+A24+A36+A80+reactivity pattern) only overlapped by four AAs (D129, R131, E154, E157). By contrast, rmAb L02 (Lin12, A1+A29+A36+A80+reactivity pattern) and rmAb E07 (A1+binding pattern) showed no overlap in their epitope footprints (FIG. 91). rmAbs E07 and 122 share overlap between their epitope footprints. Thus, the four Bmem derived rmAbs, which were representative of the four most dominant reactivity profiles, recognized unique, and in several cases, non-overlapping epitopes on A*01:01. Immune focusing of the alloreactive B cell response on the crown of HLA-A.

[0174] The data showed that the three rmAbs (E07, L02 and M07), which recognized largely non-overlapping epitopes on the top or crown of HLA A*01 :01, exhibited three distinct patterns of reactivity toward third party HLA-A alleles (FIG. 11 A-C). Since polymorphic residues in antigens may contribute to immune focusing and immunodominant B cell and ASC responses, it was predicted that binding of the rmAbs to A*01 :01 and other third-party HLA-A proteins would be dictated by the presence of one or more mismatched residues that were present in the A*01 :01 epitope recognized by the rmAb and shared by the mismatched HLA-A (A*01 :01) plus the other third party HLA-A proteins that were recognized by the same rmAb. To define the mismatched residues, A*01 :01 (donor) residues were compared to A*24:02 and A*30:01 (self) residues expressed by recipient N006 and identified 2MM residues that differed from both self-alleles as well as 1MM residues that differed between donor HLA and one of the self-HLA alleles. In agreement with the hypothesis, multiple 2MM and 1MM residues were identified that could explain the binding patterns of each of the 3 rmAbs (FIG. 11D-I). For example, the Al monospecific binding pattern of E07 (FIG. 11A) could be potentially explained by three solvent accessible residues (FIG. 111), VI 58, R163 (both 2MM residues) and D166 (1MM residue) that were present in the A*01 :01 epitope recognized by E07, were buried by or formed H-bonds with the bound E07 (R163, D166; FIG. 11E), and were not co-expressed by any of the other tested third-party class I HLA-A alleles (FIG. 11D). Thus, the data suggested that both 2MM and 1MM residues within the A* 01 :01 epitope recognized by E07 had the potential to influence the specificity and reactivity profile of the A*01:01-specific rmAbs. Importantly, these findings were not limited to the A*01:01 / E07 complex as similar results were observed when the epitopic residues and HLA binding patterns of A*01:01-specific L02 (FIG. 11F-G) and M07 (FIG. 11H-I) rmAbs were examined.

[0175] It was next asked whether conventional, non-machine learning-based epitope prediction tools would accurately identify the solvent-accessible 1MM and 2MM residues that were observed as described in the examples herein and that are contained within the structurally defined HLA epitopes recognized by the three rmAbs. Common approaches used to predict HLA residue immunogenicity as described in Kosmoliaptsis et al., Transplantation, 88: 791- 798 (2009) and Kosmoliaptsis et al., Transplantation 91 :183-190 (2011) and Duquesnoy etal., Human Immunology 78:481-488 (2017) failed to discriminate (FIG. 12A-B) between the mismatched residues that were encompassed within the epitopes (epitopic) recognized by rmAb relative to the mismatched residues that were located outside of the A*01:01 epitopes (non-epitopic). Since the standard immunogenicity tools did not accurately predict which mismatched residues were contained within the structurally defined HLA / alloAb epitope, it was asked whether the topography of HLA might influence immunogenicity and immune focusing to certain subregions of the alloantigen. It was therefore assessed whether the position of the three epitopes recognized by the four rmAbs within the quaternary structure of HLA- A*01 :01, focusing on the mismatched solvent accessible residues (defined as residues with ≥50 Å2exposed solvent accessible surface area) that could form close interactions with Ab molecules. It was found that all four rmAbs made multiple contacts (FIG. 11J and FIG. 16B) with the HLA-A a-helices (al domain residues 50-85 and a2 domain residues 138-175) that frame the peptide-binding groove (PBG) and form the membrane-distal “crown” of HLA (AH- PBG region, FIG. 8A). It was further observed that solvent-accessible residue mismatches recognized by E07, L02 and M07 (i.e., epitopic mismatches) were significantly more likely to reside within the AH-PBG on the crown of the HLA molecule as compared to mismatches that were not contained within the HLA epitopes bound by these Abs. This was true for the four solvent-accessible 2MM epitopic residues (A76 [L02], V150 [L02 and M07], V158 [E07], and R163 [E07]) (FIG. 11K) located within the AH-PBG region (p=0.0286, FIG. 11L) as well for the 9 epitopic 1MM and 2MM residues (p=0.005, FIG. 11M). Consistent with this observation, solvent-accessible 2MM residues targeted by these rmAbs were also significantly closer to the center of the PBG (p=0.0103). This was still true when both 1MM and 2MM residues (p=0.0162) were included (FIG. 11N, FIG. 12C-D) and when a lower solvent accessibility surface area threshold of 30 Å2was applied (FIG. 12E-G). Thus, these data suggest that the immunogenicity of specific 1MM and 2MM A*01:01 residues may be controlled by a combination of HLA topography and residue solvent accessibility.

[0176] AlloHLA-specific B cells target common immunodominant epitopes onA*01:01.

[0177] The data indicated that the mismatched solvent-accessible A*01:01 residues encompassed within the epitopes recognized by the three representative rmAbs were located on the crown of the HLA-A molecule within the AH-PBG region. It was predicted that the other rmAbs that were part of the same clonal lineages as E07, L02 or M07 would recognize the same “immunodominant” sites on the HLA-A a-chain. In addition, it was anticipated that rmAbs that were derived from clonal lineages unrelated to E07, L02 or M07, but exhibited the same HLA-A third-party binding patterns as the representative rmAbs, should also recognize the same immunodominant epitope(s). These postulates were tested with two of the A*01 :01-specific rmAbs, E07 and L02, which were derived from distinct clonal lineages (FIG. 6 A, 6C), exhibited different HLA reactivity patterns (FIG. 11 A-B), and recognized completely non-overlapping epitopes on A*01:01 (FIG. 91). First chimeric (c) rmAb versions of E07 and L02 were generated by fusing the IgVH and IgVt domains from the human E07 or L02 rmAbs to the mouse IgGl constant region (cE07 or cL02). Next preincubated A*01 bicoated microbeads with one or both chimeric rmAbs were used and then incubated the beads with one of the 22 test rmAbs, which included 17 A1+-monospecific human rmAbs from 8 distinct clonal lineages (FIG. 10A) and the 5 A1+A29+A36+A80+reactive human rmAbs from Lin4 and Lin12 (FIG. 10B). To measure whether the presence of the chimeric blocking Abs impeded binding by the test human rmAb, fluorochrome-labeled secondary antibodies were used specific for the constant region of human IgG, which selectively recognized the human A*01 :01-specific “test” rmAbs. For a negative control blocking Ab, a murine anti-human HLA Ab (mouse IgG2a clone W6 / 32, which was described in Barnstable et al., Cell, 14:9-20 (1978)) was used. W6 / 32 recognizes the 02m residues 3 and 89 and residue 121 in the a2 domain of the HLA a-chain (which is located outside of the AH-PBG as shown by Ladasky et al., Immunogenetics 49:312-320 (1999) and Shields et al., Tissue Antigens, 51:567-570 (1998)). W6 / 32 effectively bound to HLA-A*01:01 (FIG. 13A) but only minimally inhibited the binding of any of the A*01 :01-specific human rmAbs - with a median inhibition of only 2% (FIG. 14A). Also, the cE07 rmAb completely inhibited the binding of its non-chimeric “parent” human Lin2-derived E07 rmAb (FIG. 14A). In addition, cE07 rmAb also blocked the binding of the additional 6 Lin2-derived rmAbs (FIG. 14 A), indicating that all rmAbs in this clonal lineage likely recognized the same epitope. Moreover, cE07 rmAb almost completely inhibited (>90%) binding of 9 / 10 of the other A1+monospecific rmAbs (FIG. 14A-B), which were derived from an additional seven clonal lineages (FIG. 10 A), thereby suggesting that these genetically unrelated A1+monospecific rmAbs also likely recognized the same or a closely overlapping epitope as the A1+monospecific E07 rmAb. Consistent with the structural data showing that E07 and L02 recognize distinct, non-overlapping A*01 :01 epitopes (FIG. 91), cE07 rmAb did not greatly inhibit the binding of human Lin12 L02 rmAb or 4 other A1+A29+A36+A80+-specific rmAbs that were part of the separate Lin4 genetic lineage (FIG. 14A-B). By contrast, cL02 (an A1+A29+A36+A80+-specific rmAb) inhibited the binding of its non-chimeric “parent” Lin12 human L02 rmAb (FIG. 14A). The cL02 rmAb also blocked binding of the four A1+A29+A36+A80+-specific rmAbs from Lin4 (FIG. 14 A, 14C). By contrast, cL02 inhibited the binding of the A1+monospecific rmAbs significantly less well (FIG. 14C). Therefore, these data show that rmAbs that are either clonally related or share identical patterns of reactivity seem to recognize overlapping or neighboring epitopes on A*01:01.

[0178] While the data suggested that B cells derived from distinct clonal lineages converged on the same limited set of immunodominant HLA epitopes, it was possible that binding of a chimeric rmAb simply sterically hindered the binding of other rmAbs to non-overlapping epitopes. To test this, the five 1MM and 2MM HLA-A*01:01 residues that were present in the E07 structurally defined epitope (FIG. 11D) and were not present within the L02 structurally defined epitope were identified (FIG. 11F). From those five mismatched residues, the 2MM residues V158, R163 and the 1MM residue D166 were chosen for analysis as VI 58, R163 and DI 66 formed the closest contacts with E07, had the largest surface areas buried upon E07 binding, were located within the AH-PBG region, and formed H-bonds (R163 and D166) with E07 (FIG. HE, FIG. 13B). Three recombinant single residue mutants of A*01 :01 were generated by reverting VI 58, R163 and D166 to a “self’ HLA-A AA residue expressed by recipient N006 at each site (V158A, R163T, D166E). For example, the 2MM V158 was changed from the A*01 :01 -encoded valine to the alanine that is expressed by both self-HLA (A*24:02 and A*30:01) expressed by recipient N006. Likewise, the 1MM D166 was reverted from the donor A*01:01 aspartate to the glutamate that is expressed by recipient N006 self-HLA allele, A*30:01.

[0179] The binding activity of E07 and L02 against these A*01:01 mutants was measured by calculating the EC50 value for each rmAb against the WT (non-mutated wild-type) A*01:01 and the V158A, R163T, and D166E A*01:01 mutants (FIG. 14D-G). Consistent with the fact that all three mutant HLA featured single AA reversions within the epitope recognized by E07, E07 showed significantly decreased binding against all three A*01 :01 mutants relative to the WT A*01:01 (FIG. 14D, 14F). By contrast, L02 showed equivalent binding against the A*01 :01 mutants relative to WT A*01:01 (FIG. 14E, 14G). These results fit well with structural simulation data showing that the single point mutations in A*01:01 disrupted hydrophobic interactions between A*01:01 and the E07 H-chain (V158A, FIG. 14H) and hydrogen bonds between A*01 :01 and the E07 L-chain (R163T and DI 66E, FIG. 14I-J).To confirm these results, L-SPR was used to measure the KDof E07 and L02 for binding to WT and mutant A*01 :01. It was observed that the affinity of E07 binding to each A*01 :01 mutants was approximately 20-fold lower when compared to E07 binding to WT A*01 :01 (FIG. 13C) while the affinity of L02 binding to A*01 :01 WT was similar to that observed for binding to the A*01:01 mutants (FIG. 13D). Thus, individual mismatched A*01:01 epitopic residues, which are bound by E07 and located in the AH-PBG region, are important for A*01 :01 recognition by E07 but not L02.

[0180] Given there were single residue mutations in A*01:01 that disrupted E07 but not L02 binding to A*01:01, it was asked whether the V158A, R163T, and D166E A*01 :01 mutations affected binding of the 17 A1+monoreactive rmAbs, which were derived from 8 distinct clonal lineages (FIG. 14K-L). It was found 16 of these 17 A1+-monospecific rmAbs showed significantly decreased binding to at least one of the A*01 :01 mutants (FIG. 14K) with EC50 values that were up to 15 -fold higher against the mutant HLAs compared to the WT A*01:01 (FIG. 14L). By contrast, 4 of the 5 A1+A29+A36+A80+-specific rmAbs showed no decrease in binding to the A*01:01 mutants compared to the WT A*01:01 (FIG. 14K). To assess whether decreased binding of the rmAbs to the A*01:01 mutants correlated with cE07-mediated inhibition of binding to A* 01 :01, the 22 rmAbs were classified by the degree of inhibition by cE07 and the fold-change in the EC50 of binding to A*01:01 mutants relative to WT A*01:01. Strikingly, it was observed that rmAbs that were most inhibited by cE07 rmAb (≥50% inhibition) also exhibited higher EC50 concentrations for binding to the A*01 :01 mutants (FIG. 14M-O). Thus, >90% of the A1+monospecific rmAbs recognized an epitope in A*01 :01 that overlapped with the E07 epitope and that appeared to contain at least one of the mismatched AH-PBG-localized V158, R163, or D166 residues. These data therefore suggest that multiple genetically unrelated clones of B cells converge upon common immunodominant A* 01:01 epitopes containing solvent-accessible, exposed residues localized within the AH-PBG region.

[0181] Alloantibody and B cell responses converge on immunodominant HLA epitopes.

[0182] The data (FIG. 8-9) showed substantial connectivity between the mismatched allograft A*01:01-specific kidney and blood B cell response and the circulating A*01:01 DSA present in recipient N006. Given that immune focusing of the B cell response on specific solvent-accessible HLA-A residues within the AH-PBG region was observed, it was predicted that the circulating DSA response in recipient N006 should also be focused on these residues. To test this hypothesis, it was assessed whether the chimeric cE07 and cL02 rmAbs, which were originally cloned from the Bmem found in the kidney allograft of recipient N006, inhibited A*01 :01 binding by recipient N006 plasma-derived polyclonal Abs (FIG. 15A) or N006’s plasma-derived A* 01:01 -affinity-purified polyclonal Abs (appAbs) (FIG. 15B). It was observed that pre-incubation of A*01:01 beads with either cE07 or cL02 partially inhibited A*01 :01 binding by the total polyclonal Abs as well as the appAbs (FIG. 15A-C). Moreover, pre-incubation of A*01:01 beads with both cE07 and cL02 completely inhibited (94-99%) A*01:01 binding by the total polyclonal Abs and appAbs isolated from recipient N006 plasma (FIG. 15C). By contrast, the W6 / 32 Ab, which does not bind within the AH- PBG region, had a minimal effect on the binding of the systemic polyclonal Abs to A*01 :01 (FIG. 15C). To confirm that inhibition by cE07 was not solely due to steric hindrance, it was tested whether the A*01 :01-specific appAbs purified from the plasma of recipient N006 bound to the A*01:01 single residue mutants V158A, R163T and D166E. It was observed a 50-80% reduction in binding by the A*01:01 specific appAbs to the A*01 :01 mutants compared to WT A*01 :01 (FIG. 15D-E). Thus, the alloreactive B cell and serologic responses made by recipient N006 appeared to focus on a small number of immunodominant A*01 :01 epitopes, which contained solvent-accessible, mismatched residues within the AH- PBG region.

[0183] Shared recognition of immunodominant HLA-A epitopes across transplant recipients.

[0184] The data showed that recipient N006 made a serologic response to the A*01 :01 mismatched allograft that could be almost completely inhibited with two rmAbs, which targeted the A*01 :01 AH-PBG region and were derived from the N006’s kidney Bmem (FIG. 15F). While the AH-PBG region is comprised of 74 residues, or 27% of the entire 274 AA A*01 :01 a-chain (FIG. 15G), it was found that 73% of the 41 epitopic residues recognized by E07 and L02 were localized within the AH-PBG region (FIG. 15G). As this frequency was significantly greater (p<0.0001) than would be expected by chance, the data suggested that the topography of the HLA-A protein was critical for driving this focused B cell response. To address this idea, it was next asked whether new B cell epitope prediction models, such as GraphBepi (see Zeng et al., Bioinformatics, 39:10.1093 / bioinformatics / btadl87 (2023)) and DiscoTope-3.0 (see Hoie et al., Front Immunology, 15:1322712.

[0185] 10.3389 / fimmu.2024.1322712 (2024)) that incorporate machine learning via the quaternary protein structure prediction tool AlphaFold2, could more accurately identify these immunodominant topographically defined allo-HLA epitopes. Interestingly, both tools predicted that most of the B cell response against A*01:01 would focus on the exposed AH- PBG residues (FIG. 15F). In fact, 86% and 80% of the B cell epitopic residues predicted by GraphBepi and DiscoTope-3.0, respectively, were contained within the AH-PBG region (FIG. 15G) - a frequency that was again significantly higher (p<0.0001) than expected by chance.

[0186] The computational prediction tools supported the experimental data showing that the B cell and serologic response of recipient N006 was focused on the AH-PBG region of A*01 :01. Since the combination of the E07 and L02 rmAbs covered 41% (30 / 74) of the residues and 54% of solvent-accessible surface area in the A*01:01 AH-PBG region for recipient N006, it was hypothesized that for any given individual, more than half of any solvent-accessible 2MM residues present in the AH-PBG would be encompassed within the A*01 :01 epitopes recognized by E07 and L02. To assess this possibility, the location was mapped of all residues in a cohort of 247 HLA-A genotyped subjects who did not express A*01 :01 as one of their two self-HLA-A alleles. It was determined how many of the residues were solvent-accessible, double-mismatched between non-self (A*01:01) and the participant’s self-HLA-A alleles, localized within the AH-PBG region and contained within the A*01:01 epitopes recognized by E07 and L02 rmAbs. For most subjects, a median of five solvent-accessible 2MM AA residues that were localized within the AH-PBG region was observed (FIG. 13E). Most of these 2MM residues were also encompassed within the epitopes recognized by E07 or L02 (FIG. 15H). Indeed, for 73% (180 / 247) of the subjects in this representative cohort, 100% of the A*01:01 AH-PBG region localized, solvent- accessible 2MM residues were contained within the A*01 :01 epitopes bound by the E07 and L02 rmAbs (FIG. 15H).

[0187] The computational data supported the concept that allo-A*01 :01 specific B cell and serologic Ab responses could converge on A*01:01 epitopes that are localized within the AH-PBG region and potentially “covered” by the E07 and L02 rmAbs. If correct, it was hypothesized that chimeric cE07 and cL02 rmAbs should block binding of DSA collected from other transplant patients who generated a serologic response to a mismatched A*01 :01 allograft. It was further postulated that the inhibition mediated by cE07 and cL02 rmAbs should not be dependent on the individual’s self-HLA. To experimentally test this hypothesis, serum collected from 11 transplant recipients who received a mismatched A*01:01-typed organ and subsequently developed circulating anti-A* 01:01 DSA was examined. As expected, all 11 individuals made a robust anti-A*01:01 response to the mismatched alloantigen (FIG. 151). However, they also developed unique reactivity patterns to third-party HLA (FIG. 151). Consistent with the analysis of recipient N006 (FIG. 11L), about 62% of the 98 solvent-accessible 2MM residues in A*01 :01 that were identified across all 11 transplant recipients were located within the A*01:01 AH-PBG region, with the remaining 38% of the 2MM residues located outside of the AH-PBG region (FIG. 15 J). In addition, it was noted that the epitopes recognized by E07 and L02 encompassed every 2MM residue within the AH-PBG for 63% (7 / 11) of the subjects (FIG. 15K, FIG. 13F) - a number that was similar to the percentage calculated for the representative national cohort (FIG. 15H). Next, cE07 and cL02 rmAbs were used to test whether the circulating allospecific Ab present in these 11 A*01 :01 -mismatched recipients was focused on the AH-PBG region of A*01 :01. Strikingly, it was observed that cE07 and cL02 rmAbs alone and in combination significantly inhibited (pO.OOOl) A* 01:01 binding by circulating alloAbs from the serum of all 11 transplant recipients (FIG. 15L). Indeed, the combination of chimeric E07 and L02 rmAbs inhibited 83- 100% (mean 97%) of A* 01 :01-binding by circulating serum alloAbs present in the 11 transplant recipients (FIG. 15M). Importantly, the W6 / 32 Ab, which does not bind to the AH- PBG region, showed only 5.2% average inhibition of binding by the serum from these same recipients (FIG. 15M).

[0188] To address whether binding of the A*01 :01-specific polyclonal Abs present in these 11 transplant patient serum samples was dependent on 1MM or 2MM residues localized within the AH-PBG region, binding was compared of the subjects’ polyclonal serum alloAbs to WT A*01:01 and the 3 A*01:01 mutants V158A, R163T and D166E. Notably, even though the 11 transplant recipients expressed different combinations of self-HLA-A molecules (FIG. 151), 11 / 11 of the transplant recipients were double-mismatched at V158, 9 / 11 of the recipients were double-mismatched at R163, and 6 / 11 of the recipients were double-mismatched at DI 66 (FIG. 15N). Consistent with the hypothesis that the B cell and serologic response would be focused on the mismatched residues localized within the AH- PBG region, the serum Abs from these recipients bound less well to the 3 mutant A*01 :01 proteins relative to WT A*01:01 (FIG. 150). Indeed, while not significantly different across the entire cohort (FIG. 150), binding of the serum polyclonal Abs to V158A was reduced by an average of 32% (FIG. 15P). More strikingly, polyclonal alloAb binding to R163T and D166E was significantly decreased (FIG. 150) with an average reduction in binding of 57- 61% across the 11 individuals (FIG. 15P). When inhibition by cE07 was compared with changes in binding to the A*01 :01 mutants, it was observed that serum samples that were highly inhibited by cE07 bound significantly less well to the R163T and D166E A*01:01 mutants (FIG. 15Q). By contrast, there was no correlation between inhibition by cL02 and sensitivity to the A*01:01 mutants that targeted mismatched residues present in the epitope recognized by E07 (FIG. 15Q). Thus, the cE07 rmAb appeared to inhibit binding of polyclonal alloAbs from the 11 transplant recipients by binding to shared, specific epitopic residues located within the AH-PBG region. Taken altogether, these data suggest that the serologic response to mismatched A* 01:01 made by 12 different transplant recipients is highly focused on immunodominant epitopes that are contained within the solvent-exposed residues that are localized within the AH-PBG topographic region.

[0189]

Claims

WHAT IS CLAIMED IS:

1. One or more antibodies or modified antibodies that bind a human leukocyte antigen A*01 or A*01:01, wherein the one or more antibodies or modified antibodies block binding to at least 50% of amino acids within a face of the human leukocyte antigen A*01 or A*01:01.

2. The one or more antibodies or modified antibodies of claim 1, wherein the one or more antibodies or modified antibodies block binding to at least 75% of the amino acids within the face of the human leukocyte antigen A*01 or A*01:01.

3. The one or more antibodies or modified antibodies of claim 2, wherein the one or more antibodies or modified antibodies block binding to at least 85% of the amino acids within the face of the human leukocyte antigen A*01 or A*01:01.

4. The one or more antibodies or modified antibodies of any one of claims 1-3, wherein the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 15, 16, 54-56, 58, 62, 69, 70, 72, 73, 75-77, 79, 80, 82-84, 86-89, 106, 108, 109, 127, 129- 138, 141, 144-146, 148-151, 153-155, 157, 158, 161-163, 166, 169, 170, 173 within the face of the human leukocyte antigen A*01:01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19.

5. The one or more antibodies or modified antibodies of any one of claims 1-4, wherein the one or more antibodies or modified antibodies block binding to at least 50% of donor specific antibodies to human leukocyte antigen A* 01 or A* 01 :01 in a subject with donor specific antibodies.

6. The one or more antibodies or modified antibodies of claim 5, wherein the one or more antibodies or modified antibodies block binding to at least 75% of donor specific antibodies to human leukocyte antigen A*01 or A*01:01 in the subject with donor specific antibodies.

7. The one or more antibodies or modified antibodies of claim 6, wherein the one or more antibodies or modified antibodies block binding to at least 85% of donor specific antibodies to human leukocyte antigen A*01 of A*01:01 in a blood sample from the subject.

8. The one or more antibodies or modified antibodies of any one of claims 1-7, wherein the antibodies or modified antibodies are human.

9. The one or more antibodies or modified antibodies of any one of claims 1-8, wherein the antibodies or modified antibodies are IgGs or a portion thereof.

10. The one or more antibodies or modified antibodies of claim 5, wherein the IgGs or a portion thereof are IgGl, IgG2, IgG3, IgG4, or a portion thereof.

11. The one or more antibodies or modified antibodies of any one of claims 1-10, wherein the one or more antibodies comprise one or more full length antibodies.

12. The one or more antibodies or modified antibodies of any one of claims 1-10, wherein the one or more antibodies comprise one or more modified antibodies.

13. The one or more antibodies or modified antibodies of claim 12, wherein the one or more modified antibodies comprise one or more chimeric antibodies or portions thereof.

14. The one or more antibodies or modified antibodies of claim 12, wherein the one or more modified antibodies comprise one or more single chain antibodies or portions thereof.

15. The one or more antibodies or modified antibodies of claim 14, wherein the one or more single chain antibodies or portions thereof comprise one or more single chain variable fragments (scFvs).

16. The one or more antibodies or modified antibodies of claim 12, wherein the one or more modified antibodies or portions thereof comprise one or more Fab' fragments.

17. The one or more antibodies or modified antibodies of claim 12, wherein the one or more modified antibodies or portions thereof comprise one or more F(ab')2 fragments.

18. The one or more antibodies or modified antibodies of claim 12, wherein the one or more modified antibodies comprise one or more bispecific antibodies.

19. The one or more antibodies or modified antibodies of claim 18, wherein the one or more bispecific antibodies block binding to different amino acids within the face of the human leukocyte antigen A* 01 or A* 01 :01.

20. The one or more antibodies or modified antibodies of any one of claims 1-19, wherein the one or more antibodies or modified antibodies block binding to amino acids within the face of the human leukocyte antigen A*01.

21. The one or more antibodies or modified antibodies of any one of claims 1-19, wherein the one or more antibodies or modified antibodies block binding to amino acids within the face of the human leukocyte antigen A*01 :01.

22. The one or more antibodies or modified antibodies of any one of claims 1-21, wherein the one or more antibodies or modified antibodies comprise one or more of(a) a first antibody or portion thereof, wherein the first antibody or portion thereof comprising complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 1-6;(b) a second antibody or portion thereof, wherein the second antibody or portion thereof comprising complementarity determining regions having the amino acid sequences of SEQ ID NOs: 7-12; and(c) a third antibody of portion thereof, wherein the third antibody or portion thereof comprising complementarity determining regions having the amino acid sequences of SEQ ID NOs: 13-18.(d) a fourth antibody of portion thereof, wherein the third antibody or portion thereof comprising complementarity determining regions having the amino acid sequences of SEQ ID NOs: 312-17.

23. The one or more antibodies or modified antibodies of claims 22, wherein the one or more antibodies or modified antibodies comprise(a) a heavy chain variable region, wherein the heavy chain variable region comprises(i) a CDRH1 with an amino acid sequence comprising SEQ ID NO: 1,(ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO:2, and(iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:3; and(b) a light chain variable region wherein the light chain comprises(i) a CDRL1 with an amino acid sequence comprising SEQ ID NO:4,(ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO:5, and(iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO:6.

24. The one or more antibodies or modified antibodies of claim 23, wherein the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 54-56, 58, 62, 106, 108, 109, 129, 131, 154, 155, 157, 158, 161-163, 166, 169, 170 within the face of the human leukocyte antigen A*01:01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19.

25. The one or more antibodies or modified antibodies of claim 23 or 24, wherein the heavy chain comprises the amino acid sequence having SEQ ID NO: 20 or SEQ ID NO:26.

26. The one or more antibodies or modified antibodies of any one of claims 23-25, wherein the light chain comprises the amino acid sequence having SEQ ID NO:21 or SEQ ID NO:27.

27. The one or more antibodies or modified antibodies of claims 22, wherein the one or more antibodies or modified antibodies comprise(a) a heavy chain variable region, wherein the heavy chain variable region comprises(i) a CDRH1 with an amino acid sequence comprising SEQ ID NO:7,(ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO: 8, and(iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO:9; and(b) a light chain variable region wherein the light chain comprises(i) a CDRL1 with an amino acid sequence comprising SEQ ID NO: 10,(ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO:11, and(iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO: 12.

28. The one or more antibodies or modified antibodies of claim 27, wherein the one or more antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 15, 16, 69, 70, 72, 73, 75-77, 79, 80, 82-84, 86-89, 146, 149, 150 within the face of the human leukocyte antigen A*01 :01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19.

29. The one or more antibodies or modified antibodies of claim 27 or 28, wherein the heavy chain comprises the amino acid sequence having SEQ ID NO: 22 or SEQ ID NO:28.

30. The one or more antibodies or modified antibodies of any one of claims 27-29, wherein the light chain comprises the amino acid sequence having SEQ ID NO:23 or SEQ ID NO:29.

31. The one or more antibodies or modified antibodies of claims 22, wherein the one or more antibodies or modified antibodies comprise(a) a heavy chain variable region, wherein the heavy chain variable region comprises(i) a CDRH1 with an amino acid sequence comprising SEQ ID NO: 13,(ii) a CDRH2 with an amino acid sequence comprising SEQ ID NO: 14, and(iii) a CDRH3 with an amino acid sequence comprising SEQ ID NO: 15; and(b) a light chain variable region wherein the light chain comprises(i) a CDRL1 with an amino acid sequence comprising SEQ ID NO: 16,(ii) a CDRL2 with an amino acid sequence comprising SEQ ID NO: 17, and(iii) a CDRL3 with an amino acid sequence comprising SEQ ID NO: 18.

32. The one or more antibodies or modified antibodies of claim 31, wherein the one or more antibodies or modified antibodies block binding to at least two or more amino acids selected from the group consisting of amino acid residues 127, 129, 130, 131- 138, 141, 144-146, 148-151, 153, 154, 157 within the face of the human leukocyte antigen A*01:01, wherein the amino acid residues are numbered relative to SEQ ID NO:19.

33. The one or more antibodies or modified antibodies of claim 31 or 32, wherein the heavy chain comprises the amino acid sequence having SEQ ID NO: 24 or SEQ ID NO:30.

34. The one or more antibodies or modified antibodies of any one of claims 31-33, wherein the light chain comprises the amino acid sequence having SEQ ID NO: 25 or SEQ ID NO:31.

35. The one or more antibodies or modified antibodies of claims 22, wherein the one or more antibodies or modified antibodies comprise(c) a heavy chain variable region, wherein the heavy chain variable region comprises(iv) a CDRH1 with an amino acid sequence comprising SEQ ID NO:312,(v) a CDRH2 with an amino acid sequence comprising SEQ ID NO:313, and(vi) a CDRH3 with an amino acid sequence comprising SEQ ID NO:314 and(d) a light chain variable region wherein the light chain comprises(j) a CDRL1 with an amino acid sequence comprising SEQ ID NO:315,(jj) a CDRL2 with an amino acid sequence comprising SEQ ID NO:316, and(jjj) a CDRL3 with an amino acid sequence comprising SEQ ID NO:317.

36. The one or more antibodies or modified antibodies of claim 35, wherein the one or more antibodies or modified antibodies block binding to at least two or more aminoacids selected from the group consisting of amino acid residues 108, 109, 129, 131, 154, 157, 158, 161-163, 166, 169, 170, and 173 within the face of the human leukocyte antigen A*01 :01, wherein the amino acid residues are numbered relative to SEQ ID NO: 19.

37. The one or more antibodies or modified antibodies of claim 35 or 36, wherein the heavy chain comprises the amino acid sequence having SEQ ID NO: 318 or SEQ ID NO: 418.

38. The one or more antibodies or modified antibodies of any one of claims 35-37, wherein the light chain comprises the amino acid sequence having SEQ ID NO: 319 or SEQ ID NO: 419.

39. One or more nucleic acids encoding the one or more antibodies or modified antibodies of any one of claims 1-38.

40. A composition comprising the one or more antibodies or modified antibodies of any one of claims 1-38 and an acceptable excipient.

41. The composition of claim 40, wherein the composition is a pharmaceutical composition.

42. A kit comprising the one or more antibodies or modified antibodies of any one of claims 1-38 or the composition of claim 40 or 41.

43. The kit of claim 42, wherein the one or more antibodies or modified antibodies are attached to a solid support.

44. The kit of claim 43, wherein the one or more antibodies or modified antibodies are attached to beads.

45. The kit of claim 42, further comprising one or more human leukocyte antigen molecules.

46. The kit of claim 45, wherein the one or more human leukocyte antigen molecules are attached to a solid support.

47. The kit of claim 45, wherein the human leukocyte antigen molecules are attached to beads.

48. The kit of claim 42, further comprising one or more devices for therapeutic delivery of the one or more antibodies or modified antibodies thereof to a subject.

49. The kit of claim 41, wherein the kit is used to identify whether human leukocyte antigen A*01 or A*01:01 is expressed by cells or tissues.

50. The kit of any one of claims 42-49, wherein the one or more antibodies are in solution.

51. A method of reducing an immune response in a transplant recipient to a donor organ comprising administering to the transplant recipient the one or more antibodies or modified antibodies of any one of claims 1-38 in an amount effective to reduce the immune response in the transplant recipient as compared to a control immune response in the absence of administration of the one or more antibodies or modified antibodies.

52. The method of claim 51, further comprising screening a biological sample from the subject to identify the one or more antibodies or modified antibodies of any one of claims 1-38 that together or alone block binding of at least 50% of donor specific antibodies in the biological sample to human leukocyte antigen A*01 or A*01:01.

53. The method of claim 51 or 52, wherein the immune response comprises antibody mediated rejection of a transplanted organ or tissue.

54. The method of any one of claims 51-53, further comprising administering to the subject an immunosuppressive agent.

55. The method of any one of claims 51-54, wherein administering the one or more antibodies or modified antibodies is performed before transplantation.

56. The method of claim 54, wherein administering the immunosuppressive agent is performed before transplantation.

57. The method of any one of claims 51-56, wherein administering the one or more antibodies or modified antibodies is performed after transplantation.

58. The method of claim 56, wherein administering the immunosuppressive agent is performed after transplantation.

59. A method of treating a donor organ or tissue prior to transplantation to reduce binding of donor specific antibodies in a transplant recipient comprising contacting the donor organ with the one or more antibodies or modified antibodies of any one of claims 1- 38.

60. A method of screening a transplant recipient before or after transplantation to identify donor specific antibodies produced by the subject comprising:(a) obtaining a blood sample from the transplant recipient;(b) contacting the blood sample with one or more antibodies or modified antibodies of any one of claims 1-38; and(c) determining whether the one or more antibodies or modified antibodies block binding of at least 50% of the subject’s donor specific antibodies directed against the human leukocyte antigen A*01 or A*01:01.

61. The method of claim 60, wherein the method further comprises contacting the antibodies or modified antibodies with human leukocyte antigens A*01 or A*01:01 prior to the contacting of the blood sample followed by the determining of step (c).

62. The method of claim 60, further comprising treating the subject with the one or more antibodies or modified antibodies that block at least 50% of the subject’s donor specific antibodies.

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