EP4388012A1 - Anticorps ciblant des complexes peptidiques hla-e-hôte et leurs utilisations - Google Patents

Anticorps ciblant des complexes peptidiques hla-e-hôte et leurs utilisations

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Publication number
EP4388012A1
EP4388012A1 EP22859437.0A EP22859437A EP4388012A1 EP 4388012 A1 EP4388012 A1 EP 4388012A1 EP 22859437 A EP22859437 A EP 22859437A EP 4388012 A1 EP4388012 A1 EP 4388012A1
Authority
EP
European Patent Office
Prior art keywords
antibody
hla
cells
domain
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22859437.0A
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German (de)
English (en)
Inventor
Dapeng Li
Andrew James Mcmichael
Simon BRACKENRIDGE
Geraldine GILLESPIE
Mihai AZOITEI
Lucy C. WALTERS
Kevin O. SAUNDERS
Barton F. Haynes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Oxford
Duke University
Original Assignee
University of Oxford
Duke University
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Publication date
Priority claimed from PCT/US2021/050537 external-priority patent/WO2022060893A1/fr
Application filed by University of Oxford, Duke University filed Critical University of Oxford
Publication of EP4388012A1 publication Critical patent/EP4388012A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to antibodies that bind to HLA-E-peptide complexes thereby preventing the complex from binding to the NKG2A/CD94 heterodimeric receptor expressed on subsets of Natural Killer (NK), CD8+ T-cells, and other immune cells.
  • NK Natural Killer
  • CD8+ T-cells and other immune cells.
  • the antibodies are useful for at least NK and T-cell-based immunotherapeutic, diagnostic, and research tool strategies.
  • Killer Immunoglobulin like receptors recognize classical HLA class I molecules (Colonna and Samaridis, 1995; Karlhofer et al., 1992; Pende et al., 2019), and the NKG2A/CD94 heterodimeric inhibitory receptor interacts with the non-classical HLA class Ib molecule, HLA-E in humans and the HLA-E ortholog Qa-1b in mice. This inhibitory receptor is balanced by an activating receptor NKG2C/CD94.
  • NKG2A/CD94 is expressed on about 40 50% of peripheral blood NK cells.
  • About 5% of human peripheral blood CD8+ T cells express cell-surface NKG2A at steady state, but this expression can be upregulated by chronic antigenic stimulation.
  • NKG2A-expressing-CD8+ T-cells can form a distinct population of early activated tumor resident T cells.
  • van Montfoort et al., 2018. [0007] NKG2A is an ITIM-bearing receptor. Intracytoplasmic tyrosine based inhibitory motifs (ITIMs) are phosphorylated and recruit the phosphatases (SHP-1/2 or SHIP), which are responsible for transmitting the inhibition signal to immune effector cells.
  • ITIMs Intracytoplasmic tyrosine based inhibitory motifs
  • HLA-E has limited polymorphism with only two predominant expressed variants HLA-E*01:01 and HLA-E*01:03 that differ only in residue 107 that is outside the peptide binding groove.
  • NK cells play critical roles in immune surveillance by discriminating non-self from self, and function as effector cells by killing non-self malignant or pathogen- infected cells and producing inflammatory cytokines.
  • NK cells Specific recognition of non-self by NK cells relies on a series of inhibitory receptors, including the killer immunoglobulin-like receptor (KIR) family and the NKG2A/CD94 heterodimeric receptor.
  • KIR killer immunoglobulin-like receptor
  • HLA-E engages with NKG2A/CD94 via a restricted subset of peptides VMAPRT(L/V)(V/L/I/F)L (designated VL9) that derive from the leader sequence of HLA A, C, G and a third of B molecules.
  • HLA-E binds VL9 peptides that stabilize HLA-E surface expression (Braud et al., 1997; Braud et al., 1998.) and initiate the recognition by NKG2A/CD94 or NKG2C/CD94 on NK cells.
  • the binding affinity of the HLA-E-VL9 peptide complex is greater for NKG2A/CD94 so that the inhibitory signal dominates to suppress aberrant NK cell mediated cytotoxicity as well as cytokine production.
  • HLA-E or its murine and rhesus macaque homologs is also capable of binding to a range of other host peptides and pathogen-derived peptides, including heat shock protein 60 (Hsp60)-derived peptides (Michaelsson et al., 2002), Mycobacterium tuberculosis (Mtb) peptides (Joosten et al., 2010; van Meijgaarden et al., 2015), Simian immunodeficiency virus (SIV) Gag peptides, including the RMYNPTNIL peptide (RL9SIV) (Hansen et al., 2016) and its homolog in human immunodeficiency virus (HIV) Gag, RMYSPT
  • VL9 peptide-loaded HLA-E can protect cells from NK cell cytotoxicity.
  • the leader sequence VL9 peptides are essential not only for stabilizing HLA-E surface expression but also for determining the role of HLA-E/NKG2A/CD94 pathway in regulating NK cell self- recognition.
  • HLA-E has a broad tissue distribution, it is expressed at low surface levels in normal cells but at higher levels in tumor tissues, including melanoma and carcinomas of lung, cervix, ovarium, vulva, and head/neck (Andersson et al., 2016; de Kruijf et al., 2010; Gooden et al., 2011; Levy et al., 2008; Seliger et al., 2016; Wei and Orr, 1990) and aged cells (Pereira et al., 2019).
  • HLA-E renders tumor cells resistant to NK cell lysis (Gustafson and Ginder, 1996; Malmberg et al., 2002; Nguyen et al., 2009) and CD8+ tumor-infiltrating lymphocytes (TILs) responses (Abd Hamid et al., 2019; Eugene et al., 2019).
  • Increased HLA-E expression also contributes to the persistence of senescent cells during aging by inhibiting NK cell- and CD8+ T cell-mediated clearance (Pereira et al., 2019).
  • HLA-E expression has been reported in different tumor types (Andersson et al., 2016; de Kruijf et al., 2010; Gooden et al., 2011; Seliger et al., 2016), suggesting an inhibitory role of HLA- E/NKG2A/CD94 pathway in anti-tumor immune responses.
  • the NKG2A/CD94/HLA-E pathway is considered an important immune checkpoint target and immunotherapy strategies including antibodies targeting NKG2A have been developed.
  • the invention provides novel antibodies that can interrupt this inhibitory pathway by targeting HLA-E-peptide complexes and that have the ability to enhance NK and CD8+ T-cell effector activities.
  • the present invention provides affinity matured monoclonal antibodies (mAbs) and fragments that bind to an HLA-E- peptide complex.
  • antibody is used broadly, and can refer to a full-length antibody, a fragment, or synthetic forms.
  • the antibody binds preferentially, or specifically, to an HLA-E-VL9 peptide complex.
  • the antibody binds specifically to an HLA-E-peptide complex where the peptide is a VL9 peptide or variant thereof.
  • the peptide of said complex is a nine-mer (9 amino acids) viral peptide or a nine-mer microbiome peptide.
  • the antibody binds preferentially to a HLA-E-VL9 peptide or variant complex and is also cross-reactive to complexes presenting viral peptides or microbiome peptides.
  • the antibody can regulate the cytotoxicity effector cell function of NK and/or CD8+ T-cells positive for cell-surface expression of NKG2A (“NKG2A+”).
  • NKG2A+ NKG2A
  • monoclonal antibodies were recombinantly derived from isolated functional HLA-E-VL9- binding mAbs from HLA-E-RL9 peptide-immunized HLA-B transgenic mice and from the na ⁇ ve human B cell repertoire.
  • Such antibodies are capable of regulating effector cell cytotoxicity and can recognize HLA-E-VL9 peptide complexes expressed on the surface of tumor cells.
  • the monoclonal antibodies were affinity matured which, in some embodiments, led to enhanced VL9 peptide/HLA-E complex binding or enhanced blocking of inhibitory NKG2A binding to VL9/HLA-E complexes.
  • the affinity matured antibodies were further affinity matured which, in some embodiments, leads to enhanced VL9 peptide/HLA-E complex binding or enhanced blocking of inhibitory NKG2A binding to VL9/HLA-E complexes.
  • the further affinity matured antibodies have increased specificity to HLA-E/VL9.
  • the further affinity matured 3H4 mAbs preferentially bind to HLA-E in complex with VL-9 over HLA-E in complex with other peptides derived from viruses, such as RL9 and SARS-COV-2.
  • the invention provides methods for using affinity matured HLA-E-VL9 mAbs to modulate NK and/or CD8+T cell function as part of immunotherapeutic strategies [0015]
  • VH and VL can also be referred to as Vh or Vl and VH or VL, respectively sequences of the antibodies described in Table 1 or Figures 30A-B.
  • Table 1 may implicitly refer to Figures 30A-B that provide the nucleotide and amino acid sequences of the antibodies listed in Table 1.
  • Figures 30A-B provide nucleotide sequences for Vh and Vl domains; the amino acid sequences are readily derived from the nucleotide sequences, such as by IMGT and other online tools as cited herein, which tools not only provide amino acid translations but also predicted CDR and framework boundaries. See, e.g., http://www.imgt.org/IMGT_vquest/.
  • a nucleotide sequence for a Vh or Vl domain in Figure 30A can be input at http://www.imgt.org/IMGT_vquest/input and results include “V-REGION translation” that provides the nucleotide sequence and amino acid translation along with the framework and CDR boundaries according to the IMGT scheme.
  • the antibodies or fragments have a binding specificity that is dependent on an HLA-E-peptide complex where the peptide has an amino acid sequence according to the VL9 motif: (V/A/C/I/S/T/V/H/P)MAPRT(L/V)(V/L/I/F)L.
  • the binding specificity of the antibody or the fragment is dependent on an HLA-E- peptide complex where the peptide has an amino acid sequence according to the VL9 motif: VMAPRT(L/V)(V/L/I/F)L.
  • VL9 both motifs are referred to as “VL9” motifs, although the artisan might consider the first motif to be a VL9 variant motif.
  • the antibodies can cause an increase in cytotoxic cell numbers or activity, which can be measured by counting the number of activated cytotoxic cells in biological samples or by in vitro effector cell assays as known in the art.
  • the antibodies are recombinant antibodies having an IgG or IgM Fc domain, or a portion thereof.
  • recombinant antibodies and fragments comprising HCDR1-3 and LCDR1-3 (as used herein, the Vh CDRs can be referred to as HCDR1-3 or CDRH1-3; likewise the Vl CDRs can be referred to as LCDR1-3 or CDRL1-3) from the pairs of Vh and Vl sequences as described in Table 1 or Figures 30A-B.
  • affinity maturated antibodies lead to enhanced VL9 peptide/HLA-E complex binding or enhanced blocking of inhibitory NKG2A binding to VL9/HLA-E complexes.
  • such antibodies and fragments are humanized from a murine antibody or fragment listed in Table 1 and comprise the HCDR13 and LCDR1 3 regions from the murine antibodies or fragments.
  • the antibody comprises HCDR1-3 and LCDR1-3 of antibody 3H4_v31.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 2 or Figure 22A-E was affinity matured or further affinity matured by testing mutations in one or more of the CDRs.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 2 or Figure 22A-E was affinity matured or further affinity matured by testing mutations only in HCDR3.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 2 or Figure 22A-E was affinity matured by testing residues which contact the HLA-E-VL9 complex, including but not limited to residues outside HCDR3.
  • mutations were determined to be favorable when the antibody maintains binding specificity but improves affinity or avidity for the antigen, e.g., HLA-E-VL9.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 1 or Figures 30A-B is further affinity matured by changing one or more amino acid sequences at one or more of the CDRs.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 1 or Figures 30A-B is further affinity matured by changing one or more amino acid sequences only at HCDR3.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 1 or Figures 30A-B is further affinity matured by changing one or more amino acid sequences at one or more of the wild- type CDRs.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 1 or Figures 30A-B is further affinity matured by changing one or more amino acid sequences at HCDR3 and at one or more of the wild-type CDRs.
  • an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 1 or Figures 30A-B is affinity matured by testing residues which contact the HLA-E-VL9 complex, including but not limited to residues outside HCDR3.
  • mutations are favorable when the antibody maintains binding specificity but improves affinity or avidity for the antigen, e.g., HLA-E-VL9.
  • the invention provides optimized sequences, including without limitation affinity matured sequences or further affinity matured sequences, as described herein.
  • the optimized sequences, which are based on a murine antibody are humanized.
  • the optimized sequences comprise changes at one, two, three, four residues, or a combination of changes at any of the residues as described in the VH chain of an optimized variant 3H4 SD1, 3H4 SD2, 3H4 SD3, 3H4 SD4, 3H4 SD5, 3H4 SD6 or 3H4 SD7.
  • optimized sequences which comprise changed residues, or a combination thereof, selected from any of the residues as described in the VH chain of an optimized variant 3H4 SD1, 3H4 SD2, 3H4 SD3, 3H4 SD4, 3H4 SD5, 3H4 SD6 or 3H4 SD7.
  • the affinity matured 3H4 antibody is 3H4 Gv2, 3H4 Gv3, 3H4 Gv4, 3H4 Gv5, 3H4 Gv6, 3H4 Gv7, 3H4 Gv8, 3H4 Gv9, 3H4 Gv10, 3H4 Gv11, or 3H4 Gv12, a multimer thereof, or a fragment thereof.
  • optimized sequences which comprise changed residues, or a combination thereof, selected from any of the residues as described in the VH chain of an optimized variant 3H4 Gv2, 3H4 Gv3, 3H4 Gv4, 3H4 Gv5, 3H4 Gv6, 3H4 Gv7, 3H4 Gv8, 3H4 Gv9, 3H4 Gv10, 3H4 Gv11, or 3H4 Gv12.
  • the further affinity matured antibody is a further affinity matured optimized variant of antibody 3H4 Gv2, 3H4 Gv3, 3H4 Gv4, 3H4 Gv5, 3H4 Gv6, 3H4 Gv7, 3H4 Gv8, 3H4 Gv9, 3H4 Gv10, 3H4 Gv11, or 3H4 Gv12.
  • the further affinity matured antibody is a further affinity matured optimized variant of antibody 3H4 Gv3 (e.g., 3H4G_v31 is a further affinity matured optimized variant of antibody 3H4 Gv3).
  • the further affinity matured antibody is a further affinity matured optimized variant of antibody 3H4 Gv6 (e.g., 3H4G_v61 and 3H4G_v62 are further affinity matured optimized variants of antibody 3H4 Gv6).
  • the further affinity matured antibody is a further affinity matured optimized variant of antibody 3H4 Gv5 (e.g., 3H4G_v51 is a is a further affinity matured optimized variant of antibody 3H4 Gv5).
  • the further affinity matured 3H4 antibody is 3H4G_v31, 3H4G_v51, 3H4G_v61, 3H4G_v62, a multimer thereof, or a fragment thereof.
  • the invention provides a pharmaceutical composition comprising the recombinant antibodies of the invention.
  • the invention provides nucleic acids comprising sequences encoding anti-HLA-9-VL9 peptide complex antibodies comprising Vh and Vl sequences of the invention (e.g., the antibodies described in Table 1 or Figures 30A-B).
  • the nucleic acids are DNAs.
  • the nucleic acids are mRNAs.
  • the invention provides expression vectors comprising the nucleic acids of the invention.
  • the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive antibodies.
  • these are optionally formulated in lipid nanoparticles (LNPs).
  • the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly-A tail, 5’cap.
  • the antibodies could be administered using mRNAs without encapsulation into LNPs, particularly when applied to mucosal surfaces (Lindsay et al. Molecular Therapy Vol.28 No 3 March 2020, p.805) [0027]
  • the invention provides a kit comprising: a composition comprising an antibody of the invention, a syringe, needle, or applicator for administration of the antibody to a subject; and instructions for use.
  • the invention provides prophylactic methods comprising administering the pharmaceutical composition of the invention.
  • the invention provides methods of treatment comprising administering the pharmaceutical composition of the invention.
  • the methods are applicable to infectious diseases, malignant diseases or other conditions that would benefit from an increase in the number of stimulated effector immune cells such as NK cells and CD8+ T-cells.
  • Exemplary diseases or conditions include, but are not limited to, cancer and viral or intracellular bacterial infections.
  • the cancer comprises tumor cells that express, or overexpress HLA-E, including melanoma and carcinomas of lung, kidney, skin, prostate, stomach, rectum, cervix, ovarium, vulva, breast and head/neck.
  • Such methods of treatment can relate to methods of immunostimulation comprising the step of administering a therapeutically effective amount of an antibody of the invention, which antibody specifically binds to at least an HLA-E-VL9 peptide or variant complex and increases the number of activated NK cells or activated CD8+ cells or other cells with cytotoxic functions such as JG T-cells.
  • the therapeutic compositions and methods not only involve blocking the inhibitory HLA-E-VL9-NKG2A pathway in NK cells and CD8+ T-cells with an antibody or fragment of the invention, but also: (1) blocking other inhibitory receptors on these NK cells and CD8+ T-cells, and/or (2) promoting the activation of stimulatory receptors on these NK cells and CD8+ T-cells.
  • the targeting of multiple receptors on NK cell and CD8+ T-cell sub populations can be accomplished, for example, by the use of combination of different antibodies or agents each targeting a different receptor, or by recombinant multi-specific antibodies as described herein.
  • the administration of the anti-HLA-E-peptide complex antibodies or fragments of the invention is part of a vaccine regimen, whether the vaccine is a viral vaccine or a cancer vaccine.
  • anti-HLA-E-VL9 antibodies or fragments is part of a vaccine regimen.
  • kits for making and/or screening recombinant antibodies specific to an HLA-E-peptide complex from single circulating B-cells including steps of folding a VL9 peptide with HLA-E to make a stable complex and assembling the folded HLA-E-peptide as a tetramer, such that the labeled tetramers can be used to identify B-cells that express antibodies that specifically bind to an HLA-E-peptide of interest complex.
  • the invention provides a recombinant HLA-E-VL9 monoclonal antibody, or an antigen binding fragment thereof, which binds to an HLA-E-VL9 complex and comprises a variable heavy (Vh) domain and a variable light (Vl) domain that have amino acid sequences that have an overall 80% sequence identity to the Vh and Vl domains of an antibody listed in Table 1, or wherein the Vh domain and Vl domain each have at least 80% sequence identity to the Vh and Vl domains, respectively, of an antibody listed in Table 1 or an antibody encoded by a nucleic acid sequence in Figure 30B.
  • Vh variable heavy
  • Vl variable light
  • the antibody is an affinity matured form of 3H4 (Example 4), a fragment or a humanized version thereof.
  • the antibody is designed such that it forms hexamers.
  • the antibody is designed such that it displays full antibody or functional fragments on a nanoparticle.
  • the antibody or antigen binding fragment preferentially or specifically binds to an HLA-E-VL9 complex.
  • the Vh domain and Vl domain each have at least 90% sequence identity to the Vh and Vl domains, respectively, of an antibody listed in Table 1 or an antibody encoded by a nucleic acid sequence in Figure 30A.
  • Vl domain CDRL1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Table 1
  • Vh domain CDRH1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRH13 regions of an antibody listed in Table 1 or an antibody encoded by a nucleic acid sequence in Figure 30A.
  • the antibody or fragment is humanized or fully human.
  • the Vh domain and Vl domain of the antibody or fragment comprises framework regions that each have sufficient number of, e.g. no more than 20 or 10, amino acid variations derived from framework regions of a human antibody.
  • the framework regions are from human antibodies listed in Table 1.
  • (a) Vl domain CDRL1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Table 1
  • (b) Vh domain CDRH1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRH1-3 regions of the antibody listed in Table 1
  • (c) the Vl domain and Vh domain framework regions are derived from a human antibody.
  • the antibody or fragment is chimeric or humanized.
  • the invention provides a humanized HLA-E-VL9 monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to an HLA-E-VL9 complex and comprises: (1) a variable heavy (Vh) domain with CDRH1-3 regions derived from a murine parental antibody listed in Table 1; (2) a variable light (Vl) domain with CDRL1-3 regions derived from said murine parental antibody listed in Table 1.
  • Vh variable heavy
  • Vl variable light domain with CDRL1-3 regions derived from said murine parental antibody listed in Table 1.
  • the CDRH1-3 and CDRL1-3 regions collectively have an amino acid sequence that has no more than twenty variations as compared to the CDRH1-3 and CDRL1- 3 regions of the parental murine antibody.
  • the murine antibody listed in Table 1 is any one of the further affinity matured variants of 3H4: 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62.
  • the humanized antibodies or fragments thereof of the invention have a paratope comprising the same contact residues as 3H4, or any one of the affinity matured variants of 3H4: 3H4 Gv2, 3H4 Gv3, 3H4 Gv4, 3H4 Gv5, 3H4 Gv6, 3H4 Gv7, 3H4 Gv8, 3H4 Gv9, 3H4 Gv10, 3H4 Gv11, or 3H4 Gv12 or anyone of the further affinity matured variants of 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62.
  • the Vh domain framework regions are derived from a human antibody having a Vl domain amino acid sequence that is most similar or identical to the Vl domain amino acid sequence of the murine antibody; and wherein the Vh domain framework regions are derived from a human antibody having a Vh domain amino acid sequence that is most similar or identical to the Vh domain amino acid sequence of the murine antibody.
  • the Vh domain framework regions are derived from a human antibody having a Vh domain that has the most similar three-dimensional structure to the Vh domain of the murine antibody; and wherein the Vl domain framework regions are derived from a human antibody having a Vl domain that has the most similar three-dimensional structure to the Vl domain of the murine antibody.
  • the Vh domain framework regions are derived from IGHV3-21, IGHV3-11, IGHV3-23, IGHV1-69, or IGHV3-48.
  • the Vh domain framework region is derived from any one of the IGHV genes listed in Figure 22C.
  • the Vl domain framework regions are derived from IGKV3-15, IGKV3-20, IGKV1-39, IGKV3-11, or IGKV1-5.
  • the Vl domain framework region is derived from any one of the IGKV or IGLV genes listed in Figure 22D.
  • the binding specificity of the antibody or the fragment thereof requires the peptide of the HLA-E-VL9 complex to have an amino acid sequence according to the following motif: (V/A/C/I/S/T/V/H/P)MAPRT(L/V)(V/L/I/F)L.
  • the binding specificity of the antibody or the fragment thereof requires the peptide of the HLA-E-VL9 complex to have an amino acid sequence according to the following motif: VMAPRT(L/V)(V/L/I/F)L.
  • the antibody or fragment specifically binds to epitopes on both the HLA-E ⁇ 2 domain and the amino terminal end of the VL9 peptide.
  • the antibody, or the antigen binding fragment thereof has an affinity or avidity for the HLA-E-VL9 complex that is greater than the affinity or avidity between the HLA-E-VL9 complex and NKG2A.
  • the antibody or fragment thereof increases the cytotoxic activity of NKG2A+ NK cells, NKG2A+ CD8+ T-cells, or NKG2A+ JG T-cells, in vitro or in vivo.
  • the antibody, or the antigen-binding fragment thereof comprises an Fc moiety.
  • the antibody, or antigen-binding fragment thereof comprises a mutation(s) in the Fc moiety that reduces binding of the antibody to an Fc receptor and/or increases the half-life of the antibody.
  • the antigen binding fragment thereof is a purified antibody, a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv.
  • the antibody or antigen fragment thereof is multimerized in any suitable form. Non -limiting embodiments include hexamers formed via the Fc portion of the antibody.
  • Non-limiting embodiments include antibodies or antigen binding fragments thereof comprised in a nanoparticle. In non-limiting embodiments, these multimers increase binding avidity and/or affinity.
  • the antibody is of any isotype.
  • the invention provides an antibody or antigen binding fragment of the for use as a medicament. In certain aspect the use is in the prevention and/or treatment of a tumor comprising tumor cells that overexpress HLA-E.
  • the invention provides a nucleic acid molecule comprising a polynucleotide encoding the antibody, or the antigen-binding fragment thereof.
  • the polynucleotide sequence comprises, consists essentially of or consists of a nucleic acid sequence according to any one of the sequences in Figure 30A; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the functional variation is 80%.81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the nucleic acid is a ribonucleic acid (RNA) based on a nucleic acid or protein as shown in Figures 30A-B.
  • RNA ribonucleic acid
  • the invention provides a vector comprising a nucleic acid molecule encoding an antibody or antigen binding fragment of the invention.
  • the invention provides a cell expressing the antibody, or the antigen binding fragment of the invention; or comprising a vector comprising a nucleic acid molecule encoding an antibody or antigen binding fragment of the invention.
  • the invention provides a pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, a nucleic acid of the invention, a vector comprising a nucleic acid molecule encoding an antibody or antigen binding fragment of the invention and/or a cell expressing the antibody, or the antigen binding fragment of the invention; or comprising a vector comprising a nucleic acid molecule encoding an antibody or antigen binding fragment of the invention, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent or carrier.
  • the invention provides method of administering antibodies or antigen fragments thereof of the invention in an amount sufficient to modulation NKG2A+ NK cells or T-cells in a subject in need thereof, for example to achieve a desired therapeutic effect.
  • the invention provides methods of treating or preventing a condition that would benefit from an increase in the activation of NKG2A+ NK cells or T-cells in a subject in need thereof, comprising administering the recombinant antibody or antigen binding fragment thereof of the invention, a nucleic acid encoding these, a vector comprising a nucleic acid of the invention, or a pharmaceutical composition comprising any of these in an amount suitable to increase the number of activated cytotoxic NK cells or T-cells in the subject.
  • the methods comprise administering any combination of antibodies or antigen binding fragments of the invention. In non-limiting embodiments, the methods comprise administering any additional antibody. [0063] In certain aspects, the methods further comprise administering an additional agent that is an antagonist to an inhibitory receptor on NK cells or cytotoxic T-cells and/or an additional agent that is an agonist to a stimulatory receptor on NK cells or cytotoxic T-cells.
  • the invention provides an in vitro transcription system to synthesize ribonucleic acids (RNAs) encoding antibodies of the invention, comprising: a reaction vessel, a DNA vector template comprising nucleic acid sequence encoding an antibody of the invention as described in any of the preceding claims, and reagents for carrying out an in vitro transcription reaction that produces mRNA encoding an antibody or fragment thereof of the invention.
  • the mRNA is modified mRNA.
  • the invention provides methods for manufacturing an mRNA encoding an antibody or antigen binding fragment thereof, comprising: a.
  • an in vitro transcription reaction vessel comprising a DNA template encoding an antibody or fragment thereof according to any of the preceding claims and reagents under conditions suitable for in vitro transcription of the nucleic acid template, thereby producing an mRNA template encoding the antibody or fragment thereof according to any of the preceding claims, and b. isolating the mRNA by any suitable method of purification and separating reaction reagents, the DNA template, and/or mRNA product related impurities.
  • the mRNA comprises modified nucleotides.
  • the mRNA comprises 5’ -CAP, and/or any other suitable modification.
  • the invention provides methods manufacturing an antibody or antigen binding fragment thereof, comprising culturing a host cell comprising a nucleic acid according to any of the preceding claims under conditions suitable for expression of the antibody or fragment thereof and isolating said antibody or antigen binding fragment thereof.
  • the invention provides methods of screening for an antibody or antigen binding fragment thereof that specifically binds to an HLA-E-VL9 peptide complex, comprising: (a) providing either a substrate with immobilized HLA-E-VL9 single chain trimers or HLA-E-non-VL9 peptide control single chain trimers or with cells expressing on their surface HLA-E-VL9 single chain trimers or HLA-E-non-VL9 peptide control single chain trimers, and (b) selecting an antibody or antigen binding fragment thereof for its ability to specifically bind to the HLA-E-VL9 single chain trimers but not the HLA-E-non-VL9 peptide control single chain trimers.
  • the antigen-binding fragments are expressed on phage, and wherein said phage are incubated with the single chain trimers prior to the step of selecting an antigen binding fragment for its ability to specifically bind to the HLA-E-VL9 single chain trimers.
  • the invention provides methods for identifying a non-human antibody that specifically binds to an HLA-E-VL9 peptide complex, comprising: (a) immunizing a non human mammal with either soluble HLA E VL9 single chain trimers or with cells expressing HLA-E-VL9 single chain trimers, (b) isolating B-cells from the non- human mammal that express antibodies that can bind to HLA-E-VL9 single chain trimers but not HLA-E-non-VL9 control peptide single chain trimers.
  • certain methods further comprise recombinantly expressing the selected antibody or antigen-binding fragment and further selecting the antibody or antigen-binding fragment if it can increase the cytotoxic activity of NK cells or CD8+ T-cells when such cells are co-cultured with HLA-E-VL9 expressing cells and the antibody or antigen-binding fragment, but not when such cells are co-cultured with HLA-E- non-VL9 peptide expressing cells and the antibody or antigen-binding fragment thereof.
  • the invention provides methods for making recombinant antibodies specific to an HLA-E-peptide complex from single circulating B-cells, the method comprising: (1) folding a VL9 peptide, or other test peptide, with HLA-E to make a stable complex; (2) assembling the folded HLA-E-peptide as a tetramer; (3) staining B cells from peripheral blood of a human donor or an animal with the tetramer; (4) sorting tetramer binding B cells as single cells and cloning DNA or mRNA for antibody heavy and light chains; (4) expressing full length DNA for heavy and light chains in a suitable cell or cell-line (e.g., HEK293T) so that antibody is expressed and secreted; (5) testing specificity of the antibody or antibodies expressed and secreted from step (4) for binding to HLA-E-peptide protein complexes expressed on cells or immobilized on a substrate; and (6) purifying antibodies with requisite binding specificity,
  • the invention provides a recombinant HLA-E-VL9 monoclonal antibody, or an antigen binding fragment thereof, which binds to an HLA-E-VL9 complex and comprises: ⁇ a. Vh domain CDRH1-3 regions from an antibody listed in Table 1; and/or ⁇ Vl domain CDRL1-3 regions from an antibody listed in Table 1, wherein the Vh and Vl are from the same antibody; and ⁇ b.
  • the framework portions of the variable heavy (Vh) domain comprises amino acid sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V gene, D gene and J gene making up the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework portions of the variable light (Vl) domain comprises amino acid sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V and J genes making up the Vl gene from the corresponding antibody from which the CDRs are derived.
  • the invention provides a recombinant HLA-E-VL9 monoclonal antibody, or an antigen binding fragment thereof, which binds to an HLA-E-VL9 complex and comprises: ⁇ a. Vh domain CDRH1-3 regions from an antibody listed in Table 1; and/or ⁇ Vl domain CDRL1-3 regions from an antibody listed in Table 1, wherein the Vh and Vl are from the same antibody; and ⁇ b.
  • the framework portions of the variable heavy (Vh) domain comprises amino acid sequences that have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V gene, D gene and J gene making up the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework portions of the variable light (Vl) domain comprises amino acid sequences that have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V and J genes making up the Vl gene from the corresponding antibody from which the CDRs are derived.
  • the invention provides a recombinant HLA-E-VL9 monoclonal antibody, or an antigen binding fragment thereof, which binds to an HLA-E-VL9 complex and comprises: ⁇ a. Vh domain CDRH1-3 regions from an antibody listed in Table 1; and/or ⁇ Vl domain CDRL1-3 regions from an antibody listed in Table 1, wherein the Vh and Vl are from the same antibody; and b.
  • the framework portions of the variable heavy (Vh) domain comprises amino acid sequences that have 90% - 99% , 95%-99% sequence identity to the V gene, D gene and J gene making up the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework portions of the variable light (Vl) domain comprises amino acid sequences that have 90% 99%, 95% 99% sequence identity to the V and J genes making up the Vl gene from the corresponding antibody from which the CDRs are derived.
  • Figure 22C and 22D respectively show the immunogenetics of the Vh V/D/J genes and the immunogenetics of Vl V and J genes, along with mutation frequency of certain human antibodies. Table 5 shows the immunogenetics of mouse antibodies.
  • the CDRs are from any one of the following antibodies: 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62.
  • the immunogenetics of 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62 are as for 3H4 (see Table 5).
  • the antibody is an affinity matured 3H4 variant
  • the framework portions of the variable heavy (Vh) domain comprises, consists essentially of, consists of or has amino acid sequences derived from Vh gene 1-18 (Table 5) and wherein the framework portions of the variable light (Vl) domain comprises, consists essentially of, consists of or has amino acid sequences derived from Vk gene 14-111 (Table 5).
  • the recombinant antibody or the antigen binding fragment thereof of any of the invention is a multimer.
  • the multimer is a hexameric IgG.
  • each IgG monomer comprises a heavy chain comprising a Vh sequence and constant heavy chain sequence comprising mutations E345R, E430G and S440Y in the Fc region of a human gamma immunoglobulin gene, and a Vl sequence from the same antibody, wherein in certain embodiments the IgG is G1m3 allotype.
  • each IgG monomer comprises mutations E345R, E430G and S440Y in Fc region of human gamma immunoglobulin (G1m3 allotype) and the Vh and Vl chain are from any one of the antibodies 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62.
  • the antibody or the antigen binding fragment thereof forms a multimer displayed on a nanoparticle.
  • the nanoparticle is ferritin based nanoparticle.
  • the antigen binding fragment is a Fab fragment from any one of the antibodies 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62. ⁇ [0081] In certain embodiments, the antigen binding fragment is a Fab fragment from 3H4G_v31, 3H4G_v51, 3H4G_v61, or 3H4G_v62 antibody, wherein in certain embodiments the Fab heavy chain sequence comprises VH ( Figure 30B) immediately followed by CH1 amino acid sequence immediately followed by sortase donor sequence and wherein the Fab light chain fragment comprise 3H4VK ( Figure 30B) immediately followed by CL1 amino acid sequence.
  • the sortase donor sequence is LPXTG(G). ⁇
  • the invention encompasses the aspects described in the claims herein. BRIEF DESCRIPTION OF DRAWINGS [0084]
  • the patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color. [0085] Figures 1A-G. Isolation of monoclonal antibody 3H4 specifically targeting HLA-E-VL9 complex. (A) 3H4 bound HLA-E-VL9 single chain trimer (SCT)-transfected 293T cells.
  • SCT single chain trimer
  • All SCT constructs express EGFP to indicate transfection efficiency.
  • Transfected cells were stained with test antibody and then an Alexa fluor 555 (AF555)-anti-mouse Ig(H+L) secondary antibody.
  • AF555 Alexa fluor 555
  • a control mouse IgM TE4 was used as a negative control.
  • Anti- pan-HLA-E antibody 3D12 was used as a positive control. Representative data from one of five independent experiments are shown.
  • RL9HIV, RL9SIV, Mtb44 peptides served as peptide controls.
  • TE4 and 3D12 were used as antibody controls.
  • C-D 3H4 specifically bound to soluble HLA-E-VL9 complexes as measured by ELISA and SPR.
  • C ELISA plates were coated with 3H4 or control IgM TE4 in serial dilution, blocked, and incubated with C-trap-stabilized HLA-E-VL9, HLA-E-RL9HIV, HLA-E-RL9SIV antigens.
  • Transfected cells were stained with test antibody and then an AF647-anti-mouse Ig(H+L) secondary antibody. Isotype control stained cells were used as negative controls (grey filled histograms). Representative data from one of three independent experiments are shown.
  • (G) 3H4 recognized peptides with variants in P1.293T cells were transfected with HLA-E SCTs with VL9 peptides with single amino acid mutations at P1, then stained with 3H4 antibody followed by AF647 conjugated anti-mouse IgG(H+L) secondary antibody. Cells were gated for EGFP positive subsets.
  • FIG. 2A-J 3H4 Fab-HLA-E-VL9 co-complex structural visualisation.
  • A-B 3H4 Fab-HLA-E docking angles.
  • the HLA-E heavy chain and ⁇ 2M light chain are shown as a grey cartoon, the VL9 peptide as lime green sticks, the 3H4 HC as a light purple cartoon and the 3H4 light chain (LC) as a teal cartoon.
  • C-D Superposition of 3H4 Fab and CD94/NKG2A docking sites on HLA-E.
  • the HLA-E complex and 3H4 Fab are color-coded according to A and B.
  • the CD94 subunit is shown as an orange surface and the NKG2A subunit as a marine blue surface.
  • E Aerial view of the HLA-E-VL9 peptide binding groove surface. Non-interfacing residues of the HLA-E heavy chain are shaded light grey and non- interfacing peptide residues shaded lime green. VL9 peptide residues involved in the 3H4 interface are coloured marine blue.
  • Interfacing HLA-E HC residues that contact the 3H4 VH are shaded orange whereas those that contact the 3H4 VL are shaded teal.
  • HLA-E heavy chain residues involved in the interface with both the 3H4 VH and VL are shaded violet. Residue positions are numbered on the HLA-E surface view.
  • H-I Binding interfaces of 3H4 HC/HLA-E heavy chain (H) and 3H4 LC/HLA-E HC (I). Interfacing residues are displayed in ball-and-stick form, non-interfacing residues are displayed in cartoon form and hydrogen bonds as dashed lines.
  • interfacing residues derived from the HLA-E heavy chain include G56, S57, E58, Y59, D61, R62, E63, R65, S66 and D69 of the ⁇ 1-helix and E154, H155, A158, Y159, D162, T163 and W167 of the ⁇ 2-helix.
  • 3H4 VH (light purple) interfacing residues include N33 of the CDR1 region, N52, N54, G56 and T57 of the CDR2 region, Y97, G99, S100, S100A, Y100B and W100D of the CDR3 region and W47, D50, I58, Y59, N60, Q61 and K64 of the non-CDR VH domain.
  • HLA-E heavy chain (grey)-derived interfacing residues include E55, E58, Y59 and R62 of the ⁇ 1-helix and D162, T163, E166, W167, K170 and K174 of the ⁇ 2-helix.
  • 3H4 LC (teal) interfacing residues include Q27, D28, N30 and Y32 of the CDR1 region, D92, E93, F94 and P95 of the CDR3 region in addition to D1 of the VL domain.
  • J Key interfacing residues within the germline-encoded D-junction.3H4 HC amino acid sequence with the VH segment in purple and the CDR1/2/3 regions shaded grey.
  • Germline-encoded residues within the VH CDR3 D-junction are denoted.
  • the 4 key interfacing residues (Y97, S100, S100A and Y100B) within this germline-encoded D-junction that make contacts with both the HLA-E heavy chain and VL9 peptide are highlighted magenta in the sequence and illustrated as magenta sticks in the PyMol visualisation.
  • the HLA-E heavy chain and VL9 peptide are displayed as grey and green cartoons, respectively, with key interfacing residues in stick form. Hydrogen bonds are depicted as magenta dashed lines and residues of the 3H4 VH domain that are not germline encoded key interfacing residues are displayed in light purple cartoon form.
  • FIGS 3A-E show that MAb 3H4 enhanced the cytotoxicity of the NKG2A+ NK cell line NK-92 against HLA-E-VL9 expressing 293T cells.
  • A Schematic illustrating the hypothesis. Blockade of the inhibitory NKG2A/CD94/HLA-E pathway with anti-HLA-E-VL9 antibody (3H4) and/or anti-NKG2A antibody (Z199) could enhance target cell lysis by NK cells.
  • B-C NK cell cytotoxicity against 3H4 IgM-treated target cells as assessed by 51Cr release assay.
  • Antibody was incubated with HLA-E-VL9 transfected 293T cells (B) and untransfected 293T cells (C) at final concentration of 10 ⁇ g/ml or 3 ⁇ g/ml, and NK92 cells were added into the mixture as effector cells at effector: target (E:T) ratios of 20:1 and 6:1.
  • Mouse IgM MM-30 was used as an isotype control. Dots represent the mean values of triplicate wells in eight independent experiments. Statistical analysis was performed using mixed effects models. Asterisks show the statistical significance between indicated groups: ns, not significant, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG. 1 Schematic illustration of the affinity maturation strategy.
  • Libraries of 3H4 mAb variants were transformed into S. cerevisiae and displayed on the surface of yeast cells as single-chain fragment variable (scFv).
  • scFv single-chain fragment variable
  • APC-conjugated HLA-E-VL9 tetramers were used for FACS sorting.
  • B Sites at the 3H4/HLA-E-VL9 interface where sequence optimization by library screening provided the most significant affinity gains.3H4: purple; HLA-E: green; VL9 peptide: orange. See Figures 6A-C for VH sequences.
  • C Enrichment of HLA-E-VL9+ library clones after three rounds of selection by fluorescence- activated cell sorting (FACS).
  • the yeast cells containing the scFv libraries were sorted sequentially for binding to decreasing concentrations of fluorescently labeled HLA-E-VL-9 (50 ⁇ g/ml, top; 10 ⁇ g/ml, middle; or 0.6 ⁇ g/ml bottom).
  • D Mutations at positions 97-100 in the eleven 3H4 variants chosen for additional characterization upon library screening.
  • E Binding of 3H4 Gwt and optimized variants to HLA E VL9 or HLA E Mtb44 transfected 293T cells. Representative flow cytometry data from one of three independent experiments are shown.
  • F SPR sensorgrams showing binding kinetics of 3H4 Gwt and optimized variants.
  • Rate constants (ka, kd) and dissociation constant KD were determined by curve fitting analysis of SPR data with a 1:1 binding model. Binding data are shown as black lines, and the best fits of a 1:1 binding model are shown as colored lines. Representative data from one of two independent experiments are shown.
  • G Enhanced NK-92 cell cytotoxicity by optimized IgG 3H4 Gv3 and 3H4 Gv6 on HLA-E-VL9 transfected 293T cells and untransfected 293T cells, in compare with IgG 3H4 Gwt. Dots represent the mean values of triplicate wells in four or five independent 51Cr release assays. Statistical analysis was performed using mixed effects models.
  • FIG. 5A-L HLA-E-VL9-specific antibodies exist in the B cell pool of healthy humans.
  • A Scheme of isolating HLA-E-VL9-specific antibodies from healthy humans. Pan- B cells were first isolated by negative selection from human leukapheresis PBMCs. A three- color sorting strategy was used to sort single B cells that were positive for HLA-E-VL9 and negative for HLA-E-RL9HIV or HLA-E-RL9SIV.
  • HLA-E-VL9 double positive, HLA-E-RL9HIV negative, HLA-E-RL9SIV negative B cells in PBMCs from four donors are shown.
  • Variable regions of antibody heavy and light chain genes were isolated from the sorted B cells by PCR and cloned into an expression backbone with a human IgG1 constant region.
  • Antibodies were produced by transient transfection in 293i cells, and antibody binding specificities were analyzed by surface staining of transfected 293T cells and high throughput screening (HTS) flow cytometry.
  • (B) Binding specificities of the HLA-E-VL9-specific antibodies (n 56) from four donors shown as a heatmap. The compensated MFIs of HLA-E-VL9-specific antibody staining on HLA-E-VL9, HLA-E-RL9HIV, or HLA-E-RL9SIV transfected 293T cells at a concentration of 1 ⁇ g/ml were shown. Representative data from one of two independent experiments are shown.
  • Human antibody CA147 was incubated with HLA-E-VL9 transfected 293T cells and untransfected 293T cells at final concentration of 10 ⁇ g/ml or 3 ⁇ g/ml, and NK92 cells were added into the mixture as effector cells at effector: target (E:T) ratio of 20:1 and 6:1.
  • Human antibody A32 was used as the isotype control. Dots represent the mean values of triplicate wells in five independent experiments. Statistical analysis was performed using mixed effects models. Asterisks show the statistical significance between indicated groups: ns, not significant, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • (E) Phenotypes of HLA-E-VL9-specific B cells (n 56) shown as heatmap. Expression of markers in each single B cell were determined from index sorting data and are shown as MFIs after compensation. Compensated MFIs below zero were set as zero. Each row indicates one single cell. The rows were clustered by K-means Clustering in R.
  • CD10-CD27-CD38+/- na ⁇ ve B cells CD10+CD27-CD38++ transitional B cells, CD10-CD27+CD38- non-class-switched memory B cells, and CD10-CD27+CD38+ plasmablast cells.
  • Figure 22E Detailed information for each single cell and antibody is shown in Figure 22E.
  • F-G Antibody gene usages.
  • F Heavy chain viable (VH) region gene usage shown as a bar chart (left) and pie chart (right). The top five VH genes found in HLA-E-VL9-specific antibodies are colored in the pie charts.
  • V ⁇ Kappa chain variable
  • V ⁇ lambda chain variable
  • V ⁇ region gene usage shown as a bar chart (left) and pie chart (right).
  • the top five V ⁇ /V ⁇ genes found in HLA-E-VL9-specific antibodies are colored in the pie charts.
  • H-I Comparison of heavy chain (H) and light chain (I) CDR3 (CDR-H3) length.
  • HLA-E-VL9 antibody CDR-H3 length was compared with the reference (DeKosky et al., 2015) human antibody CDR-H3 length.
  • J-K Violin plots showing the mutation rates of heavy chains (J) and light chains (K).
  • L Mouse or human HLA-E-VL9-specific antibodies cross-react with microbiome-derived peptides presented by HLA-E.
  • FIG. 6A C shows a summary of affinity matured antibodies (A), non limiting embodiments of nucleic acids (B) and amino acids (C). In this figure, underlined are the four positions changed relative to the original 3H4 VH sequence.
  • Figure 6C discloses SEQ ID NOS 35-45, respectively, in order of appearance.
  • Figures 7A-K provide the nucleotide sequences of the Vh and Vl domains for antibodies listed in Table 1.
  • the CDR regions are underlined according to IMGT convention, which is only one embodiment of possible CDR boundaries.
  • Figures 8A-H provide the amino acid sequences of the Vh and Vl domains for antibodies listed in Table 2.
  • the CDR regions are underlined according to IMGT convention, which is only one embodiment of possible CDR boundaries.
  • Figure 8A discloses SEQ ID NOS 128-135, respectively, in order of appearance.
  • Figure 8B discloses SEQ ID NOS 136- 143, respectively, in order of appearance.
  • Figure 8C discloses SEQ ID NOS 144-151, respectively, in order of appearance.
  • Figure 8D discloses SEQ ID NOS 152-159, respectively, in order of appearance.
  • Figure 8E discloses SEQ ID NOS 156, 141, 160-164, and 157, respectively, in order of appearance.
  • Figure 8F discloses SEQ ID NOS 165-167, 157, and 168-171, respectively, in order of appearance.
  • Figure 8G discloses SEQ ID NOS 172-179, respectively, in order of appearance.
  • Figure 8H discloses SEQ ID NOS 180-187, respectively, in order of appearance.
  • Figure 9. A selection scheme of optimized libraries to affinity mature antibodies.
  • Figure 10. Antibody residues to be optimized through site directed libraries mapped on the structure of the 3H4-HLA-E/VL9 complex.
  • Antibody residues showed in spheres and displayed in the same color will be varied at the same time in the respective site directed libraries.
  • 3H4 antibody is shown in purple (light chain) and magenta (light chain).
  • HLA-E is shown in green and the VLP peptide in orange.
  • Figure 11 Non-limiting embodiments of scFv variants (3H4 SD1-7) comprising contact residues which could be modified in an optimized variant. Based on structural analysis, seven scFv different directed libraries that sample all the amino acid variants at four sites were designed. The four sites are shown in bold and red in the sequence.
  • A-B Expression of human HLA-B27 and ⁇ 2M in peripheral blood lymphocytes (PBLs) of the transgenic (TG) mice.
  • HLA-B27/ ⁇ 2M TG mice were used to minimize the induction of antibodies to HLA class I and ⁇ 2M.
  • FIG. 1 Schematic diagram of the immunization, splenocyte fusion and hybridoma screening strategy.
  • SCT single-chain trimer
  • Antigens used for immunizations and ELISA assays are all cysteine (C)-trap stabilized. Each curve represents one animal, and the curve for animal that we used for splenocyte fusion are shown in red.
  • C cysteine
  • E Affinity of 3H4 binding to soluble HLA-E-VL9 complex.3H4 as a mouse IgM or as a recombinant human IgG1 were immobilized on CM5 sensor chips and soluble HLA-E-VL9 complex protein at the indicated concentrations was flowed over antibody immobilized sensor chips. Binding data are shown as black lines, and the best fits of a 1:1 binding model are shown as colored lines.
  • Rate constants (ka, kd) and dissociation constant KD were measured following curve fitting analysis.
  • F Purification of 3H4 by FPLC using Superose 6 size exclusion column. The arrowed peak was collected and analyzed by negative staining.
  • G Representative class average images of 3H4 negative stain electron microscopy (NSEM).
  • H Binding of 3H4 expressed in a human backbone G1.4A to a panel of autoantigens by AtheNA assays. HIV-1 gp41 antibody 4E10 was set as a positive control, and a Flu antibody Ab82 was used as a negative control. The dotted lines indicate the cutoff values ⁇ 100 luminance units used to denote positivity.
  • Transfected cells were stained with 3H4 antibody or an anti- ⁇ 2M control antibody 2M2 followed by AF647 conjugated anti-mouse IgG(H+L) secondary antibody. Data are representative from one of three independent experiments.
  • 3H4 recognizes peptides with variants in P1.293T cells were transfected with HLA-E SCTs with VL9 peptides with single amino acid mutations at P1, then stained with 3H4 antibody or an anti- ⁇ 2M control antibody 2M2 followed by AF647 conjugated anti-mouse IgG(H+L) secondary antibody (dark blue). Cells were gated for EGFP positive subsets.
  • FIG. 13 Phenotypic analysis of NK-92 cells. Related to Figure 3. NKG2A and CD94 expression in NK-92 cell line detected by flow cytometry. NK-92 cells were stained with PE-CD94 antibody or FITC-NKG2A antibody and analyzed in flow cytometer. A PE- isotype and FITC-isotype antibodies were used as negative controls. [0098] Figure 14. Related to Figure 3.
  • NK-92 cells were stained with BV650-CD16 antibody, APC-CD32 antibody, or BV421-CD64 antibody and analyzed in flow cytometer.
  • Peripheral blood mononuclear cells (PBMCs) were used as positive controls. Dot plots overlay of antibody stained cells (red) and unstained control cells (grey) were shown.
  • NK-92 cells were negative for CD16, CD32 or CD64, while a subset of PBMC cells were positive for each antibody. Data from a single antibody phenotype experiment.
  • Figures 15A C Crystallographic data for the 3H4 Fab and VL9 bound HLA E co complex structure.
  • C Table of RMSD (root mean square deviation) in ⁇ between chains of the 3H4-HLA-E-VL9 co-complex structure. Two copies of the 3H4 Fab-HLA-E-VL9 co- complex were present in the asymmetric unit and thus RMSD between chains related by non- crystallographic symmetry was calculated via C ⁇ atom pairwise alignment on the PDBePISA server. Average C ⁇ atom RMSD following pairwise alignment is also reported for the HLA- E heavy chain (HC) of 1MHE (Chain A), a previously published non-receptor-bound VL9- loaded HLA-E complex, and the HLA-E HC from the 3H4-HLA-E-VL9 structure reported here (Chain A).
  • Figure 16 Crystallographic data for the 3H4 Fab and VL9-bound HLA-E co- complex structure. Related to Figure 3. Table listing residues involved in the interface between the 3H4 VH and the VL9 peptide. [0101]
  • Figures 17A-D Crystallographic data for the 3H4 Fab and VL9-bound HLA-E co- complex structure. Related to Figure 3. (A) Table of interacting residues of the 3H4 VH and HLA-E HC interface. (B) Table of interacting residues of the 3H4 VL and HLA-E HC interface. (C-D) Hydrogen bonding and salt bridges in the 3H4-HLA-E-VL9 structure.
  • Amino acid atom abbreviations: C - mainchain Carbon atom, O - mainchain Oxygen atom, N - mainchain Nitrogen atom, CA - ⁇ -Carbon atom, CB - ⁇ -Carbon atom, CD - ⁇ -Carbon atom, CE - ⁇ -Carbon atom, CG - ⁇ -Carbon atom, CH - ⁇ -Carbon atom, CZ - ⁇ -Carbon atom, OD - ⁇ -Oxygen atom, OE - ⁇ -Oxygen atom, OG - ⁇ -Oxygen atom, OH - Oxygen atom, ND ⁇ Nitrogen atom, NE ⁇ Nitrogen atom, NH Nitrogen atom, NZ ⁇ -Nitrogen atom.
  • Figures 18A-E Affinity optimization of 3H4 IgG.
  • FIG. 1 NK cell cytotoxicity against wild-type 3H4 IgG-treated target cells measured by 51Cr release assay. Unoptimized, wild-type 3H4 on a human IgG1 backbone (3H4 Gwt) was incubated with HLA-E-VL9 transfected 293T cells and untransfected 293T cells at final concentration of 10 ⁇ g/ml, 1 ⁇ g/ml, or 0.1 ⁇ g/ml, and NK92 cells were added into the mixture as effector cells at effector: target (E:T) ratios of 20:1 and 6:1.
  • Wild-type 3H4 and variants were titrated on HLA-E-VL9-transfected or untranfected 293T cells. Mean fluorescent intensity (MFI) from one of three independent experiments were shown.
  • FIG. 20A-J Isolation and characterization of human HLA-E-VL9-specific antibodies. Related to Figure 5.
  • A-B HLA-E-VL9-specific B cell phenotyping from mice.
  • Splenic cells isolated from HLA-B27/ ⁇ 2M TG mice (A) or C57BL/6 mice (B) were stained and analysed flow cytometry for B cells (B220+CD19+) that are HLA-E-VL9 double positive, HLA-E-RL9HIV negative and HLA-E-RL9SIV negative.
  • C Gating strategy of the single cell sorting for HLA E VL9 specific B cells from a Cytomegalovirus (CMV) negative, male human.
  • CMV Cytomegalovirus
  • the enriched cells were stained and gated on viable/CD14neg/CD16neg/CD3neg/CD235aneg/CD19pos/HLA-E-VL9pos/HLA-E- RL9HIVneg/ HLA-E-RL9SIVneg subset as shown.
  • Cells were single-cell sorted into 96-well plates for the downstream PCR cloning. Representative data from one of the four donors were shown.
  • D-E Flow cytometry titration of purified HLA-E-VL9-specific mAbs isolated from a CMV-negative, male human.
  • Antibodies recovered from sorted B cells were constructed in human IgG1 backbones and used for staining titration on both C-trap- stabilized and unstabilized HLA-E-VL9, HLA-E-RL9SIV, HLA-E-RL9HIV transfected 293T cells. EGFP expression indicates transfection efficiency. Transfected cells were stained with testing antibodies at the concentration of 2 ⁇ g/ml, followed by secondary antibody AF555-anti-human IgG staining.
  • D Staining data of a representative antibody CA147 and a negative control antibody CA136.
  • E Summary of the MFI of antibody binding data shown as bar chart. Data are representative from one of two independent experiments.
  • MFI of the indicated antibody staining on wildtype VL9 peptide was set as 100%, and the percentages equals to (MFI of binding on each P1 variant) / (MFI of binding on wildtype VL9) x 100%.
  • H Affinity measurements of human HLA-E-VL9 antibodies binding to soluble HLA-E-VL9 complex. Human antibodies CA123 or CA147 on human IgG1 backbone was immobilized on CM5 sensor chips and soluble HLA-E-VL9 complex protein at the indicated concentrations was flowed over the antibody immobilized sensor chips. Rate constants (ka, kd) and dissociation constant KD were measured following curve fitting analysis.
  • HLA-E-VL9-specific antibodies Gene usage and cross-reactivities of the HLA-E-VL9-specific antibodies. Related to Figure 5.
  • A-B Heavy chain (JH) gene usage shown as bar chart (A) and pie chart (B).
  • C-D Kappa chain (Jk) and lambda chain (JL) gene usage shown as bar chart (C) and pie chart (D).
  • E Human microbiome- derived peptides bound and stabilized HLA-E as indicated by upregulated HLA-E expression.
  • microbiome-derived peptides were loaded on K562 cells, and stained with HLA-E antibody 3D12 or isotype control mouse IgG at the concentration of 10 ⁇ g/ml. No peptide loaded cells were used as the negative control, and mtb44 peptide-loaded cells were set as the positive control. Data are representative from one of three independent experiments. (F-G) 3H4, CA143 and CA147 cross-reacted with human microbiome-derived peptides presented by HLA-E.
  • HLA-E binders as shown in panel A were loaded on K562 cells, and stained with mouse antibodies (3H4 or isotype control mouse IgM TE4; panel F), as well as human antibodies (CA136, CA143 or CA147; panel G) at the concentration of 10 ⁇ g/ml. No peptide loaded cells were used as the negative control, and mtb44 peptide-loaded cells were set as the positive control. Data are representative from one of three independent experiments. [0106] Figures 22A-E. HLA-E-VL9-specific antibodies isolated from humans. Figures 22A and B provide Mean Fluorescent Intensity data from binding specificity experiments.
  • Figures 22C and D provides for each antibody information on: Vh V-gene, D-gene, and J-gene family information; Vl V-gene and J-gene family information; CDR3 lengths; isotype; and mutation frequency information.
  • the % mutation frequency is calculated from the mutation frequency in column U.
  • Figure 22E provide a correlation between the SEQ ID in the concurrently filed sequence listing for the nucleotide sequence for the Vh and Vl domains for each antibody.
  • VL9-like peptides tested in this study a VL9 peptide sequence was first searched by similarity in NIH Microbial database, and resultant microbial sequences were then analyzed using an MHC binding prediction tool for human HLA-E binding and mouse Qa1 binding (http://tools.immuneepitope.org/analyze/html/mhc_binding.html). Peptides that fulfilled the following criteria were selected for downstream validation experiments: 1) related to human pathogens, 2) 9 aa in length, and 3) with HLA-E binding score above 0.2. Related to Figure 5.
  • Figures 24A and B show non-limiting embodiments of nucleic acid and amino acid sequences of antibodies (3H4 and CA147) designed to form hexamer. Any of the antibodies described herein could be designed to form hexamers. Non-limiting embodiments include any of the affinity matured antibodies described herein.
  • Figure 24B discloses SEQ ID NOS 337- 341, respectively, in order of appearance.
  • Figures 25A-C shows expression of hexameric IgG forms of anti-HLA-E antibodies. (A) location of Fc mutations that generate IgG hexamers.
  • FIG. 26A-C Two-dimensional class averages of negative stain electron microscopy images of (B) 3H4 or (C) CA147 IgG hexamers. Arrows indicate the antigen-binding fragment (Fab) and crystallizable fragment (Fc).
  • Figures 26A-C shows non-limiting embodiments of nucleic acid and amino acid sequences of antibody or fragment thereof sequences designed to form ferritin nanoparticles, or hexamers.
  • Figure 26A shows Amino acid sequence of full-length immunoglobulin genes (signal peptide is underlined; sortase acceptor sequence GGGGGSG is italicized). In Figure 26A, signal peptide is underlined.
  • Figure 26A discloses SEQ ID NOS 342-345, 348-352, 350, 353, and 344, respectively, in order of appearance.
  • Figure 26B shows Vh and Vl sequences of various antibodies.
  • Figure 26B shows amino acid sequences of only the variable regions of 3H4, CA147.
  • Figure 26B discloses SEQ ID NOS 128, 129, 184, 185, 184, 185, 128, and 129 respectively, in order of appearance.
  • Figure 26C shows non-limiting embodiments of nucleic acid sequencing encoding amino acids in Figures 26A-B. Any of the antibodies described herein could be designed to form nanoparticles and/or hexamers. Non-limiting embodiments include any of the affinity matured antibodies described herein.
  • Figures 27A-B shows negative stain electron microscopy images of (A) CA117 or (B) CA147 Fab nanoparticles. A and B. Raw images of Fab nanoparticles. The inset in B shows 3-D class average of the nanoparticle with Fab arrayed around the nanoparticle surface.
  • Figure 28 shows that CA147 Fab nanoparticles recognize VL9:HLA-E complexes on the cell surface. See Example 3 for nanoparticle description.
  • FIG. 29A-B shows data concerning CA147 IgG with mutations within the Fc region form hexamers.
  • CA147 IgG1 with three mutations (designated CA147_G1m3, with three mutations E345R/E430G/S440Y) were produced by transfected 293i and purified by Protein L IgK light chain beads.
  • FIGS. 30A-B show the amino acid sequences of the Vh and Vl domains for antibodies listed in Table 1.
  • the CDR regions are underlined according to IMGT convention, which is only one embodiment of possible CDR boundaries.
  • Figure 30A discloses SEQ ID NOS 388-392, respectively, in order of appearance.
  • Figure 30B discloses SEQ ID NOS 405- 409, respectively, in order of appearance.
  • Figure 31 shows a mouse tumor study protocol.
  • Figure 32 shows tumor volume (mm 3 ) over time for Group 1 (3H4_Gv3 + NK92 + IL-2), Group 2 (CA147_G1.4A + NK92 + IL2), Group 3 (CH65 + NK92 + IL-2), and Group 4 (CH65 + IL-2).
  • Figure 33 shows mouse survival curves for Group 1 (3H4_Gv31_G1.4A + NK92 + IL-2), Group 2 (CA147_G1.4A + NK92 + IL2), Group 3 (CH65_G1.4A + NK92 + IL-2), and Group 4 (CH65_G1.4A + IL-2). * indicates p ⁇ 0.005.
  • Figure 34 shows tumor volume (mm 3 ) over time for Group 1 (3H4_Gv31 + NK92 + IL-2), Group 2 (CH65 + NK92 + IL-2), Group 3 (3H4_Gv31 + IL-2), Group 4 (3H4_Gv31 + NK92), Group 5 (3H4_Gv31), and Group 6 (CH65).
  • Figure 35 shows mouse survival curves for Group 1 (3H4_Gv31 + NK92 + IL-2), Group 2 (CH65 + NK92 + IL-2), Group 3 (3H4_Gv31 + IL-2), Group 4 (3H4_Gv31 + NK92), Group 5 (3H4_Gv31), and Group 6 (CH65).
  • Figure 36 shows HLA-E-VL9 binding kinetics to either original/isolated 3H4 IgM or IgG.
  • Figure 37 shows a schematic diagram of HLA-E-VL9 binding to immobilized 1 st generation optimized 3H4 IgG mAbs.
  • Figure 38 shows HLA-E-VL9 kinetics on 3H4 IgG variants: 3H4G_v3, 3H4G_v6, and 3H4G_v7.
  • Figure 39 shows a repeat study of HLA-E-VL9 kinetics on 3H4G variants: 3H4G_V3, 3H4G_v6, and 3H4_CDRH3_7HC.
  • Figure 40 shows a schematic diagram of HLA-E-VL9 binding to immobilized 2 nd generation optimized 3H4 IgG mAbs.
  • Figure 41 shows HLA-E-VL9 kinetics on captured 3H4 IgG variants: 3H4G_v31, 3H4G_v51, 3H4G_v61, and 3H4G_v62.
  • Figure 42 shows binding specificity of affinity matured 3H4 mAbs.
  • the present invention relates to antibodies and antigen binding fragments thereof, including recombinant and/or derivative forms, that bind to HLA-E-peptide complexes.
  • the antibodies or fragments bind to epitopes on the HLA-E-VL9 peptide complex.
  • the antibodies or fragments can have a binding specificity that is sensitive to the presence of a VL9 peptide as presented by HLA-E.
  • the art defines the VL9 peptide as having a nine amino acid motif according to the following formula: VMAPRT(L/V)(V/L/I/F)L.
  • the antibodies of the invention specifically bind an HLA-E-VL9 complex where the peptide has an amino acid sequence according to the formula: VMAPRT(L/V)(V/L/I/F)L
  • the antibodies of the invention bind an HLA E VL9 complex where the peptide has an amino acid sequence according to the following formula: (V/A/C/I/S/T/V/H/P)MAPRT(L/V)(V/L/I/F)L.
  • the peptide has an amino acid sequence variation at position P2 according to the following as a VL9 peptide or a VL9 variant.
  • Non limiting embodiments of VL-9 peptides are listed in Table 4.
  • the antibodies can prevent intercellular signaling between HLA-E expressing cells and NKG2A expressing cells, i.e., the HLA-E- NKG2A pathway, which inhibits cytotoxic effector cell functions.
  • the antibodies are useful in conditions where diseased or infected cells express HLA-E-VL9 complexes (or HLA-E-peptide complexes with peptides that do not confirm to the VL9 motifs recited above, including viral peptides and microbiome peptides, where an HLA-E- VL9 specific antibody of the invention is cross-reactive to the HLA-E-peptide complex) and where the condition would benefit from an increase in effector cell function against these cells.
  • Recombinant antibodies of the invention include antibodies derived from rearranged VDJ variable heavy chain (Vh) and/or rearranged VJ variable light chain (Vl) sequences from individual or clonal cells that express an antibody that specifically binds to HLA-E-VL9 (or other HLA-E-peptide complex of interest), and optionally is further able to prevent or inhibit binding between the HLA-E-VL9 complex and the NKG2A/CD94 heterodimeric complex.
  • Vh VDJ variable heavy chain
  • Vl VJ variable light chain
  • the antibody is cross-reactive to HLA-E-peptide complexes where the peptide does not conform to VL9 motifs but where this HLA-E-non-VL9 peptide complex still engages with the NKG2A/CD94 complex such as with certain viral peptides and microbiome peptides.
  • Antibodies are described in the accompanying examples, figures, and tables, and the invention includes antibodies comprising CDR sequences contained with the Vh and Vl amino acid sequences described herein.
  • the invention provides monoclonal antibodies.
  • the monoclonal antibodies are produced by a clone of B lymphocytes.
  • the monoclonal antibody is recombinant and is produced by a host cell into which an expression vector(s) encoding the antibody, or fragment thereof, has been transfected.
  • Methods for obtaining rearranged heavy and light chain sequences are well known in the art and often involve amplification-based-cloning and sequencing. Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies or antibody fragments of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
  • PCR polymerase chain reaction
  • the invention encompasses antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75% identical to the Vh and/or Vl variable domain amino acid sequences of the antibodies described herein in the Figures or Table 1. Further, the invention encompasses variants having one or mutations (99% et seq.
  • the variant maintains antigen binding specificity to the HLA-E-VL9 complex, and in some embodiments, maintains the ability to specifically bind an epitope that includes the part of ⁇ - 2 domain of HLA-E and the amino terminal end of the VL9 peptide, (2) the variant does not have a decrease in binding affinity or avidity that is more than 10-fold, 5-fold, 2-fold, or 1- fold than the corresponding antibody of the Figures or Table 1, (3) the variant has a binding affinity or avidity that is an improvement of more than 100-fold, 10-fold, 5-fold, 2-fold, or 1- fold more than the corresponding antibody of the Figures or Table 1, (4) the variant does not have a decrease in promoting cytotoxic activity by NK cells or CD8+ cells that is more than 10-fold, 5-fold, 2-fold, or 1-fold as compared to the corresponding antibody of the Figures or Table 1, (5) the variant has an increase in in in
  • binding specificity can be determined by any suitable assay in the art, for example but not limited competition binding assays, epitope mapping, etc.
  • epitope mapping can be conducted by using cells expressing HLA-E single chain trimers (SCTs) presenting different peptides including VL9 peptides with single amino acid mutations. The cells are incubated with an antibody to be tested and stained with secondary antibodies.
  • SCTs HLA-E single chain trimers
  • Binding specificity is measured by counting the number of positively-stained cells using flow cytometry where specific binding to an HLA-E-peptide complex of interest is shown by differences in the number of positively stained cells as compared to experiments using cells that express HLA-E in complex with control and/or mutant peptides.
  • binding specificity can be determined by testing whether the antibody can bind to cells pulsed with a peptide of interest and that express HLA-E and is HLA class I negative; but not to the same cells pulsed with a negative control peptide or mutant peptides with sequences that differ from the peptide of interest.
  • antibody 3H4 was found to be able to preferentially bind HLA-E-peptide complexes with peptide variants of the classical VL9 motif, i.e., able to bind to complexes with peptides mutated at position 1 to alanine, cysteine, isoleucine, serine, threonine, valine, histidine, proline; but not to peptides mutated at position 1 to arginine, glutamate, glycine, lysine, methionine, asparagine, tryptophan, tyrosine, or phenylalanine.
  • binding specificity can be determined in the context of epitope mapping where a peptide of interest is mutated and loaded into HLA-E complexes to test for differences in binding.
  • an antibody or fragment of the invention has a binding specificity characterized by its ability to preferentially bind to an HLA-E-VL9 complex, where the peptide conforms to a VL9 motif as used herein or a variant thereof, but is not able to bind to a control HLA-E-peptide complex where the peptide is the RL9HIV peptide or the RL9SIV peptide.
  • an antibody or fragment preferentially binds to an HLA-E-viral peptide complex. In some embodiments, an antibody or fragment preferentially binds to an HLA-E-microbiome complex. [0138] Another binding specificity assay can test whether the antibody can specifically bind to soluble HLA-E-peptide complexes using ELISA or SPR (or HLA-E-peptide complexes are immobilized and the antibody is soluble).
  • HLA-E-VL9 peptides were used.
  • Control antibodies that bind to HLA-E but are not specific to the HLA-E-VL9 complex are known in the art, for example, the pan-HLA-E mAb 3D12.
  • Another binding specificity assay can test whether the antibody can specifically bind to peptides that can form a complex with HLA-E by analyzing FACS. (See, e.g., Example 7, where HLA-E, RL9HIV R1V, SARS-CoV-2001, Mtb and Mamu-E peptides were used.)
  • Affinity can be measured, for example, by surface plasmon resonance.
  • SPR affinity measurements can provide the affinity constant K D of an antibody, which is based on the association rate constant k on divided by the disassociation rate constant k off .
  • comparing affinity between a variant and an antibody of Table 1 is based on K D .
  • the comparison is based only on k off .
  • the antibodies should have the same valency, i.e., Fab vs. Fab, scFv vs. scFv, IgG v. IgG, IgM v. IgM, etc.
  • affinity is a measure of functional affinity.
  • functional affinity covers the binding strength of a bi- or polyvalent antibody to antigens that present more than one copy of an epitope, because they are multimeric or conjugated in multiple copies to a solid phase, thus allowing cross-linking by the antibody.
  • a monovalent antibody fragment e.g., Fab
  • SPR often immobilizes antigen on a solid substrate and the antibody is flowed over the substrate thereby allowing kinetic measurements of antibody association and disassociation rates).
  • Avidity can also be measured by SPR.
  • Avidity can be quantitatively expressed, for example, by the ratio of K D for a Fab over the multivalent form, e.g., IgG, IgM et al.
  • Potency can be measured, for example, by a NK cell or CD8+ T-cell cytotoxicity assay as known in the art.
  • the cytotoxicity assay utilizes different HLA-E-peptide complex expressing cells as the target cells.
  • Target cells are incubated with antibodies and NKG2A+ NK cells or NKG2A+ CD8+ T-cells (effector cells) and pulsed with 51 Cr (chromium).
  • the invention provides antibodies with CDR amino acid sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the CDR1, 2, and/or 3 of Vh (also referred to as CDRH1, CDRH2, and CDRH3) and/or CDR1, 2, and/or 3 of Vl (also referred to as CDRL1, CDRL2, and CDRL3) amino acid sequences of the antibodies of Table 1 or the amino acid sequences translated from the nucleotide sequences of the antibodies of Figure 30A.
  • the invention provides antibodies with CDR amino acid sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to CDRs to an antibody of Table 1 or the amino acid sequences translated from the nucleotide sequences of the antibodies of Figure 30A, where each CDR can have a different percent identity.
  • the antibody has at least 99%, 98%, 97%, 96%, or 95% identity for all CDRs as compared to the CDRs of an antibody listed in Table 1 or Figures 30A-B except HCDR3 and LCDR3, which can allow for a lower percent identity, for example, 99% to 80%, 99% to 85%, 99% to 90%, or 99% to 95%.
  • the invention provides antibodies which can tolerate a larger percent variation in the sequences outside of the Vh and/Vl sequences of the antibodies.
  • the invention provides antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65% identical, wherein the identity is outside of the Vh or Vl regions, or the CDRs of the Vh or Vl chains of the antibodies described herein.
  • the antibody or antigen binding fragment thereof comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the sequences in Figure 30B, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the functional variation is 80%.81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the person of ordinary skill in the art can select from one or more CDR conventions to identify the boundaries of the CDR regions.
  • the antibody or antigen binding fragment thereof comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the sequences in Figure30B, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the antibody or antigen binding fragment thereof comprises framework regions corresponding to the heavy and light chain gene usage of the HLA-E-VL9-specific antibodies described herein.
  • human antibodies see Figure 22C, columns R, S and T, 22D columns AA and AB
  • mouse antibodies see Table 5 for non-limiting embodiments of gene usage for human and mouse antibodies respectively.
  • the heavy chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to VH gene 3-21, 3- 23, 3-48, 3-74, 3-11, 4-30-2, 3-30, 1-18, 1-69, 4-61, 3-30-3, 3-15, or 4-59.
  • the light chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to a kappa light chain.
  • the light chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to a lambda light chain.
  • the light chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to VL gene 3-21, 1-44, 2-14, 5-39, 2-8, or 2-23 or VK gene 3-20, 1-39, 1-16, 3-15, 1-33, 2-28, 3-11, 1-9, 2-14, 1-5, 1-8, 4-1, 1D-12, 6-21, 1-17, or 14-111.
  • the antibody or antigen binding fragment thereof comprises framework regions corresponding to the heavy and light chain gene and allele usage of the HLA-E-VL9-specific antibodies described herein.
  • the heavy chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to the *01, *04, *18, *05, *03, *06, or *12 allele of VH gene.
  • the light chain of the antibody or antigen binding fragment thereof comprises a framework region corresponding to the *01, *02, or *03 allele of the VL or VK gene.
  • the antibody or antigen binding fragment thereof comprises, consists essentially of or consists of a Vh amino acid sequence or a Vl amino acid sequence in Figure 30B or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the functional variation is 80%.81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a Vh amino acid sequence and/or a Vl amino acid sequence according to Figure 30B.
  • the invention provides antibodies that were affinity matured in vitro.
  • the affinity of an antibody to its antigen target can be modulated by identifying mutations introduced into the variable region generally or into targeted sub-regions. For example, it is known in the art that one can sequentially introduce mutations through each of the CDRs, optimizing one at a time, or to focus on CDRH3 and CDRL3, or CDRH3 alone, because it often forms the majority of antigen contacts.
  • Biol.215:403, 1990 is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • sequence identity is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
  • CDRs and Frameworks are defined according to the IMGT scheme.
  • IMGT-defined CDR regions have been highlighted/underlined in the nucleotide and amino acid sequences for each of the Vh and Vl variable regions of the antibodies of Table 1.
  • IMGT sequence analysis tools will identify CDR and framework regions in the nucleotide sequence and translated amino acid sequence. See http://www.imgt.org/IMGT_vquest/analysis [0155]
  • CDR and framework regions can be identified based on other classical variable region numbering and definition schemes or conventions, including the Kabat, Chothia, Martin, and Aho schemes.
  • the ANARCI Antigen receptor Numbering And Receptor Classification; see http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/) online tool allows one to input amino acid sequences and to select an output with the IMGT, Kabat, Chothia, Martin, or AHo numbering scheme. With these numbering schemes, CDR and framework regions within the amino acid sequence can be identified. The person of ordinary skill is able to ascertain CDR and framework boundaries using one or more of several publicly available tools and guides.
  • Table 3A provides a general, not limiting guide, for the CDR regions as based on different numbering schemes (see http://www.bioinf.org.uk/abs/info.html#cdrid). In the Table, any of the numbering schemes can be used for these CDR definitions, except the Contact CDR definition uses the Chothia or Martin (Enhanced Chothia) numbering. [0157] Table 3A. General Guide of Selected CDR Definitions [0158] In Table 3A above, for CDRH1 Kabat numbering using the Chothia CDR definition, the boundary is H26 to H32 or H34 because the Kabat numbering convention varies between H32 and H34 depending on the length of the loop.
  • FIG. 30A-B some CDR boundaries are underlined, and these boundaries are defined according to the IMGT scheme.
  • framework (FR) regions constitute all of the variable domain sequence outside of the CDRs, once CDR boundaries are identified, framework regions are necessarily identified. The convention within the art is to label the framework regions as FR1 (sequence before CDR1), FR2 (sequence between CDR1 and CDR2), FR3 (sequence between CDR2 and CDR3), and FR4 (sequence after CDR3).
  • FR1 sequence before CDR1
  • FR2 sequence between CDR1 and CDR2
  • FR3 sequence between CDR2 and CDR3
  • FR4 sequence after CDR3
  • the ABnum tool numbers the amino acid sequences of variable domains according to a large and regularly updated database called Abysis, which takes into account insertions of variable lengths and integrates sequences from Kabat, IMGT, and the PDB databases.
  • the Honneger scheme is based on structural alignments of the 3D structures of immunoglobulin variable regions and allows one to define structurally conserved CD positions and deduction of appropriate framework regions and CDR lengths (Honegger and Plückthun, J. Mol. Biol., 2001, 309:657-70).
  • Ofran et al. used a multiple structural alignment approach to identify the antigen binding residues of the variable regions called “Antigen Binding Regions (ABRs)” (Ofran et al., J.
  • ABRs Antigen Binding Regions
  • ABRs can be identified using the Paratome online tool that identifies ABR by comparing the antibody sequence with a set of antibody–antigen structural complexes (Kunik et al., Nucleic Acids Res., 2012, 40:521-4).
  • Another alternative tool is the proABC software, which estimates the probabilities for each residue to form an interaction with the antigen (marieri et al., Bioinformatics, 2013, 29:2285-91).
  • the CDRs of the antibodies of the invention are defined by the scheme or tool that provides the broadest or longest CDR sequence.
  • the CDRs are defined by a combination of schemes or tools that provides the broadest/longest CDRs. For example, from the Table of CDR Definitions above, CDRL1 would be L24-L36, CDRL2 would be L46-L56, CDR3 would be L89-L97, CDRH1 would be H26-H35/H35B, CDRH2 would be H47-H65, and CDRH3 would be H93-H102.
  • the CDRs are defined by the Anticalign software, which automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences from an algorithm based on rules from the Kabat and Chothia conventions (Jarasch et al., Proteins Struct.
  • the CDRs are defined by a combination of the Kabat, IMGT, and Chothia CDR definitions. In some embodiments, the CDRs are defined by the Martin scheme in combination with the Kabat and IMGT schemes. In some embodiments, the CDRs are defined by a combination of the Martin and Honneger schemes. In some embodiments, the CDRs comprise the ABR residues identified by the Paratome tool. [0165] The complete human immunoglobulin germline gene loci and alleles are available in the Immunogenetics Database (IMGT).
  • IMGT Immunogenetics Database
  • the invention provides antibody fragments, which have the binding specificity and/or properties of the inventive antibodies. Recombinant fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains.
  • Antibody "fragments" include Fab, Fab', F(ab')2, F(ab)c, diabodies, Dabs, nanobodies, and Fv fragments.
  • heavy or light chain monomers and dimers single domain heavy chain antibodies, single domain light chain antibodies, (a single domain antibody, sdAb, is also referred to in the art as a nanobody) as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.
  • sdAb single domain antibody
  • single chain Fv single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.
  • a cleavage site can be included in a linker, such as a furin cleavage site.
  • a recombinant antibody can also comprise a heavy chain variable domain from one antibody and a light chain variable domain from a different antibody.
  • the invention encompasses chimeric antigen receptors (CARs; chimeric T cell receptors) engineered from the variable domains of antibodies. (Chow et al, Adv. Exp. Biol. Med., 2012, 746:121-41).
  • the Chimeric Antigen Receptor consists of an antibody-derived targeting domain (including fragments such as scFv or Fab) fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR.
  • Fc Domains [0171] Whether full-length, or fragments engineered to have a Fc domain, (or particular constant domain portions thereof), the antibodies of the invention can be of any isotype or have any Fc (or portion thereof) of any isotype. It is well-known in the art how to engineer Fc domains or portions together with antibody fragments.
  • the antibodies of the invention can be used as IgG1, IgG2, IgG3, IgG4, whole IgG1 or IgG3s, whole monomeric IgAs, dimeric IgAs, secretory IgAs, IgMs as monomeric, pentameric, hexameric, or other polymer forms of IgM.
  • the class of an antibody comprising the VH and VL chains described herein can be specifically switched to a different class of antibody by methods known in the art.
  • the nucleic acid encoding the VH and VL can encode an Fc domain (immunoadhesin).
  • the Fc domain can be an IgA, IgM or IgG Fc domain.
  • the Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No.20100093979, incorporated herein by reference.
  • the immunoadhesin is an IgG1 Fc.
  • the immunoadhesin is an IgG3 Fc.
  • the IgG constant region comprises the LS mutation. Additional variants of the Fc portion of the antibody are also contemplated by the invention. See Maeda et al. MAbs.2017 Jul; 9(5): 844–853. Published online 2017 Apr 7, PMID: 28387635; see also Booth et al.
  • the antibodies comprise amino acid alterations, or combinations thereof, for example in the Fc region outside of epitope binding, which alterations can improve their properties.
  • Fc modifications are known in the art.
  • the invention contemplates antibodies comprising mutations that affect neonatal Fc receptor (FcRn) binding, antibody half-life, and localization and persistence of antibodies at mucosal sites. See e.g. Ko SY et al., Nature 514: 642-45, 2014, at Figure 1a and citations therein; Kuo, T.
  • the antibodies comprise AAAA substitution in and around the Fc region of the antibody that has been reported to enhance ADCC via NK cells (AAA mutations) containing the Fc region aa of S298A as well as E333A and K334A (Shields RI et al JBC, 276: 6591-6604, 2001) and the 4 th A (N434A) is to enhance FcR neonatal mediated transport of the IgG to mucosal sites (Shields RI et al. ibid).
  • Other antibody mutations have been reported to improve antibody half-life or function or both and can be incorporated in sequences of the antibodies.
  • modifications such as but not limited to antibody fucosylation, may affect interaction with Fc receptors (See e.g. Moldt, et al. JVI 86(11): 66189-6196, 2012).
  • the antibodies can comprise modifications, for example but not limited to glycosylation, which reduce or eliminate polyreactivity of an antibody. See e.g. Chuang, et al. Protein Science 24: 1019-1030, 2015.
  • the antibodies can comprise modifications in the Fc domain such that the Fc domain exhibits, as compared to an unmodified Fc domain enhanced antibody dependent cell mediated cytotoxicity (ADCC); increased binding to Fc ⁇ RIIA or to Fc ⁇ RIIIA; decreased binding to Fc ⁇ RIIB; or increased binding to Fc ⁇ RIIB. See e.g. US Pub 20140328836.
  • ADCC antibody dependent cell mediated cytotoxicity
  • the invention provides a multivalent and multispecific antibody.
  • a multivalent antibody has at least two antigen-binding sites, i.e., at least two heavy/light chain pairs, or fragments thereof.
  • Antibody fragments may impart monovalent or multivalent interactions and be contained in a variety of structures as described above.
  • monovalent scFv molecules may be synthesized to create a bivalent diabody, a trivalent "triabody” or a tetravalent "tetrabody.”
  • the scFv molecules may include a domain of the Fc region resulting in bivalent minibodies.
  • sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets.
  • Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126 1136).
  • multivalent but not multispecific antibodies are provided, where the multispecific antibody comprises multiple identical Vh/Vl pairs, or the CDRs from the Vh and a Vl pairs. This type of multispecific antibody will serve to improve the avidity of an antibody.
  • a tetramer can comprise four identical scFvs where the scFv is based on the Vh/Vl pair from an antibody of Table 1.
  • multivalent but not multispecific antibodies comprise multiple Vh/Vl pairs (or the CDRs from the pairs) where each pair binds to an overlapping epitope. Determining overlapping epitopes can be conducted, for example, by structural analysis of the antibodies and competitive binding assays as known in the art. [0185] In some embodiments, multispecific antibodies comprise multiple Vh/Vl pairs (or the CDRs from the pairs) where each pair binds to a distinct epitope (not overlapping) on the HLA-E-VL9 complex.
  • multispecific antibodies or fragments of the invention comprise at least a Vh and a Vl pair from Table 1, or the CDRs from the Vh and a Vl pair, in order to provide the multispecific antibody with binding specificity to the HLA-E-VL9 peptide complex.
  • the multispecific antibody can have one or more additional binding specificities by further comprising antibody binding site fragments from antibodies that bind to different antigens.
  • a multispecific antibody can comprise a Vh/Vl pair that targets the HLA-E-VL9 peptide complex from Table 1 and one or more Vh/Vl pairs that target and block different inhibitory receptors.
  • inhibitory receptors include, but are not limited to, NKG2A, CD94, NKG2A/CD94 heterodimer, LAG- 3, TIM-3, TIGIT, BTLA, PD-1, and CTLA-4.
  • a multispecific antibody can comprise a Vh/Vl pair that targets the HLA-E-VL9 peptide complex from Table 1 and one or more Vh/Vl pairs that specifically bind and operate as agonists upon stimulatory receptors for effector cell function.
  • Non-limiting examples of stimulatory receptors for effector cell function include NKG2C, NKG2D, 4-1BB (CD137), OX40 (CD134), TNFRSF7 (CD27), ICOS (CD278), TNFRSF8 (CD30), LFA-2 (CD2), DNAM-1 (CD226), CD3, CD16, CD32, and CD64.
  • the invention provides a bispecific antibody.
  • a bispecific or bifunctional/dual targeting antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites (see, e.g., Romain Rouet & Daniel Christ “Bispecific antibodies with native chain structure” Nature Biotechnology 32, 136–137 (2014); Garber “Bispecific antibodies rise again” Nature Reviews Drug Discovery 13, 799– 801 (2014), Figure 1a; Byrne et al. “A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications” Trends in Biotechnology, Volume 31, Issue 11, November 2013, Pages 621–632 Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315- 321 (1990); Kostelny et al., J.
  • the bispecific antibody is a whole antibody of any isotype. In other embodiments it is a bispecific fragment, for example but not limited to F(ab)2 fragment. In some embodiments, the bispecific antibodies do not include Fc portion, which makes these diabodies relatively small in size and easy to penetrate tissues.
  • Non-limiting examples of multispecific antibodies also include: (1) a dual-variable- domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so called “dock and lock” molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragment
  • bispecific antibodies examples include but are not limited to BiTE (Micromet), DART (MacroGenics) (e.g., US Patents 8,795,667; US Publications 20090060910; 20100174053), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • the multispecific antibodies can include a Fc region.
  • Fc bearing DARTs are heavier, and could bind neonatal Fc receptor, increasing their circulating half-life.
  • the invention encompasses multispecific molecules comprising an Fc domain or portion thereof (e.g. a CH2 domain, or CH3 domain).
  • the Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM.
  • the Fc domain (or portion thereof) is derived from IgG.
  • the IgG isotype is IgG1, IgG2, IgG3 or IgG4 or an allotype thereof.
  • the multispecific molecule comprises an Fc domain, which Fc domain comprises a CH2 domain and CH3 domain independently selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2 domain derived from IgG and the CH3 domain derived from IgE, or the CH2 domain derived from IgG1 and the CH3 domain derived from IgG2, etc.).
  • the Fc domain may be engineered into a polypeptide chain comprising the multispecific molecule of the invention in any position relative to other domains or portions of the polypeptide chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the Vl and Vh domains of the polypeptide of the chain; may be n-terminal to both the Vl and Vh domains; or may be N-terminal to one domain and c-terminal to another (i.e., between two domains of the polypeptide chain)).
  • the present invention also encompasses molecules comprising a hinge domain.
  • the hinge domain be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM.
  • the hinge domain is derived from IgG, wherein the IgG isotype is IgG1, IgG2, IgG3 or IgG4, or an allotype thereof.
  • the hinge domain may be engineered into a polypeptide chain comprising the multispecific molecule together with an Fc domain such that the multispecific molecule comprises a hinge-Fc domain.
  • the hinge and Fc domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein.
  • the hinge and Fc domain are separated by at least one other domain of the polypeptide chain, e.g., the Vl domain.
  • the hinge domain, or optionally the hinge-Fc domain may be engineered into a polypeptide of the invention in any position relative to other domains or portions of the polypeptide chain.
  • a polypeptide chain of the invention comprises a hinge domain, which hinge domain is at the C-terminus of the polypeptide chain, wherein the polypeptide chain does not comprise an Fc domain.
  • a polypeptide chain of the invention comprises a hinge-Fc domain, which hinge-Fc domain is at the C- terminus of the polypeptide chain.
  • a polypeptide chain of the invention comprises a hinge Fc domain, which hinge Fc domain is at the N terminus of the polypeptide chain.
  • the invention encompasses multimers of polypeptide chains, each of which polypeptide chains comprise a Vh and a Vl domain, comprising CDRs as described herein.
  • the Vl and Vh domains comprising each polypeptide chain have the same specificity, and the multimer molecule is bivalent and monospecific.
  • the Vl and Vh domains comprising each polypeptide chain have differing specificity and the multimer is bivalent and bispecific.
  • the polypeptide chains in multimers further comprise an Fc domain.
  • Fc domains Dimerization of the Fc domains leads to formation of a diabody molecule that exhibits immunoglobulin-like functionality, i.e., Fc mediated function (e.g., Fc-Fc ⁇ R interaction, complement binding, etc.).
  • Fc mediated function e.g., Fc-Fc ⁇ R interaction, complement binding, etc.
  • staphylococcus Sortase A transpeptidase ligation to conjugate antibodies or fragments, for e.g. but not limited to a nanoparticle.
  • a C-terminal LPXTG(G) tag or a N-terminal pentaglycine repeat tag is added to the gene encoding antibody or fragment thereof, where X signifies any amino acid, such as Ala, Ser, Glu.
  • a nanoparticle carrying the complementary tag is provided.
  • Sortase A is then used to covalently bond the tagged antibody or fragment thereof to a nanoparticle.
  • the sortase A-tagged antibody or fragment thereof can also be conjugated to other peptides, proteins, or fluorescent labels.
  • the sortase A tagged antibody or fragment thereof are conjugated to ferritin to form nanoparticles.
  • ferritin is H. pylori ferritin. Any suitable ferritin can be used.
  • ferritin sequences are disclosed in WO/2018/005558.
  • sequences herein include c-terminal sortase A donor sequences to allow for site specific conjugation to multimerizing scaffolds expressing the n-terminal sortase A acceptor sequence.
  • the donor sequence is a LPXTGG where the third amino acid can vary.
  • X is E.
  • the acceptor sequence is composed of 5 or more glycines appended to the N-terminus.
  • Any suitable ferritin can be used in the nanoparticles of the invention.
  • the ferritin is derived from Helicobacter pylori. In non-limiting embodiments, the ferritin is insect ferritin. In non limiting embodiments, each nanoparticle displays 24 copies of the spike protein on its surface. [0198] Another non-limiting approach to multimerize antibodies or fragments uses mutations E345R, E430G, S440Y in the Fc region of human gamma immunoglobulin (G1m3 allotype) that allow formation of hexamers (see Diebolder, CA et al Science 343:1260-632014).
  • diabody molecules of the invention encompass tetramers of polypeptide chains, each of which polypeptide chain comprises a Vh and Vl domain.
  • two polypeptide chains of the tetramer further comprise an Fc domain.
  • the tetramer is therefore comprised of two ⁇ heavier ⁇ polypeptide chains, each comprising a Vl, Vh and Fc domain, and two ⁇ lighter ⁇ polypeptide chains, comprising a Vl and Vh domain. Interaction of a heavier and lighter chain into a bivalent monomer coupled with dimerization of the monomers via the Fc domains of the heavier chains will lead to formation of a tetravalent immunoglobulin-like molecule.
  • the monomers are the same, and the tetravalent diabody molecule is monospecific or bispecific. In other aspects the monomers are different, and the tetravalent molecule is bispecific or tetraspecific.
  • Formation of a tetraspecific diabody molecule as described supra requires the interaction of four differing polypeptide chains. Such interactions are difficult to achieve with efficiency within a single cell recombinant production system, due to the many variants of potential chain mispairings.
  • One solution to increase the probability of mispairings is to engineer "knobs-into-holes" type mutations into the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization.
  • an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a ⁇ knob ⁇ , e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., ⁇ the hole ⁇ (e.g., a substitution with glycine).
  • Such sets of mutations can be engineered into any pair of polypeptides comprising the diabody molecule, and further, engineered into any portion of the polypeptides’ chains of the pair.
  • the invention also encompasses diabody molecules comprising variant Fc or variant hinge-Fc domains (or portion thereof), which variant Fc domain comprises at least one amino acid modification (e.g. substitution, insertion deletion) relative to a comparable wild-type Fc domain or hinge-Fc domain (or portion thereof).
  • Molecules comprising variant Fc domains or hinge-Fc domains (or portion thereof) normally have altered phenotypes relative to molecules comprising wild-type Fc domains or hinge-Fc domains or portions thereof.
  • the variant phenotype may be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function as assayed in an NK dependent or macrophage dependent assay.
  • Fc domain variants identified as altering effector function are known in the art. For example International Application WO04/063351, U.S. Patent Application Publications 2005/0037000 and 2005/0064514.
  • the bispecific diabodies of the invention can simultaneously bind two separate and distinct epitopes.
  • the two separate epitopes are on different cells, e.g., HLA-E-VL9 epitope on one cell and a stimulatory receptor epitope on another cell. In certain embodiments, the two separate epitopes are on two different inhibitory receptors on the same cell. In certain embodiments the epitopes are from the same antigen. In other embodiments, the epitopes are from different antigens. In some embodiments, at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g. CD3, CD16, CD32, CD64, etc.) which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells.
  • an immune effector cell e.g. CD3, CD16, CD32, CD64, etc.
  • the diabody molecule binds to the effector cell determinant and also activates the effector cell.
  • diabody molecules of the invention may exhibit Ig-like functionality independent of whether they further comprise an Fc domain (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g., ADCC assay).
  • the bispecific antibodies engage cells for Antibody- Dependent Cell-mediated Cytotoxicity (ADCC).
  • the bispecific antibodies engage natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages.
  • the bispecific antibodies are T-cell engagers.
  • the bispecific antibody comprises an HLA-E-VL9 binding fragment and a CD3 binding fragment.
  • Various CD3 antibodies are known in the art. See for example US Patent 8,784,821. The CD3 antibodies may be activating or non-activating to recruit CD8 T cells as effector cells (See for e.g. Sung et al. J Clin Invest.2015;125(11):4077–4090. https://doi.org/10.1172/JCI82314).
  • the bispecific antibody comprises a HLA-E-VL9 binding fragment and CD16 binding fragment.
  • the invention provides antibodies with dual targeting specificity.
  • the invention provides bi-specific molecules that are capable of localizing an immune effector cell to an HLA-E over-expressing cell, such as a tumor cell or a virally infected cell, so as facilitate the killing of this cell.
  • bispecific antibodies bind with one "arm” to a surface antigen on target cells, and with the second "arm” to an activating, invariant component of the T cell receptor (TCR) complex or to an activating, invariant component of a different stimulatory receptor such as NKG2C on NK cells or other immune effector cells.
  • the simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and effector cell, causing, for example, activation of any cytotoxic T cell or NK cell and subsequent lysis of the target cell.
  • the immune response is re-directed to the target cells and may be independent of classical MHC class I peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs.
  • bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.
  • BiTE bispecific T cell engager
  • Several bispecific antibody formats have been developed and their suitability for T cell mediated immunotherapy investigated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promise in the clinic (reviewed in Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)).
  • BiTEs are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker.
  • DART dual affinity retargeting
  • DART dual affinity retargeting molecules are based on the diabody format that separates cognate variable domains of heavy and light chains of the two antigen binding specificities on two separate polypeptide chains but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)).
  • the invention also contemplates Fc-bearing DARTs.
  • triomabs which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical trials, represent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).
  • the invention also contemplates bispecific molecules with enhanced pharmacokinetic properties. In some embodiments, such molecules are expected to have increased serum half- life. In some embodiments, these are Fc-bearing DARTs (see supra).
  • bispecific molecules comprise one portion which targets HLA-E-VL9 and a second portion which binds a second target. In certain embodiments, the first portion comprises Vh and Vl sequences, or CDRs from the antibodies described herein.
  • the second target could be, for example but not limited to an effector cell.
  • the second portion is a T-cell engager.
  • the second portion comprises a sequence/paratope which targets CD3, CD16, or another suitable target.
  • the second portion is an antigen-binding region derived from a CD3 antibody, optionally a known CD3 antibody.
  • the anti-CD antibody induce T cell-mediated or NK-mediated killing.
  • the bispecific antibodies are whole antibodies.
  • the dual targeting antibodies consist essentially of Fab fragments.
  • the dual targeting antibodies comprise a heavy chain constant region.
  • the bispecific antibody does not comprise Fc region.
  • the bispecific antibodies have improved effector function. In certain embodiments, the bispecific antibodies have improved cell killing activity.
  • Various methods and platforms for design of bispecific antibodies are known in the art. See for example US Pub.20140206846, US Pub. 20140170149, US Pub.20090060910, US Pub 20130295121, US Pub.20140099318, US Pub.20140088295 which contents are herein incorporated by reference in their entirety.
  • the invention also provides trispecific antibodies comprising binding specificities of the invention antibodies. Non-limiting embodiments of trispecific format is described in Xu et al. Science 06 Oct 2017, Vol.358, Issue 6359, pp.85-90.
  • the invention also provides CAR T cell designs which comprise antigen binding portions or fragments incorporating portions of Vh and Vl sequences as described herein.
  • Chimeric and Humanized Antibodies include chimeric and humanized forms of non-human Vh and Vl sequences, or portions thereof.
  • chimeric and humanized antibodies can be based on the murine antibodies listed in Table 1.
  • the CDR regions based on the IMGT convention are underlined.
  • antibody variable regions of the Fab is a major focus of engineering because of its role in antigen or target binding.
  • the antigen combining site is formed by the combination of the six CDR or hypervariable regions, three from the heavy chain and three from the light chain.
  • Chimeric antibodies are well-known in the art and have a design where the non- human Vh and Vl variable domain sequences are spliced together with human heavy chain and light chain constant domain sequences.
  • humanized antibodies are created by combining at the genetic level (engineering, grafting), the CDR regions of a non-human antibody (usually murine) with the framework sequences of a human antibody variable domain.
  • many of the humanization techniques involve engineering one or more of the six non-human antibody CDRs onto different templates or frameworks. In some embodiments of the invention, all six CDRs are grafted, in others just CDRH3 and CDRL3.
  • the humanized antibody comprises Vl domain framework regions that are derived from a human antibody having a Vl domain amino acid sequence that is most similar or identical to the Vl domain amino acid sequence of the murine antibody, and wherein the Vh domain framework regions are derived from a human antibody having a Vh domain amino acid sequence that is most similar or identical to the Vh domain amino acid sequence of the murine antibody.
  • the humanized antibody comprises Vh domain framework regions are derived from a human antibody having a Vh domain that has the most similar three-dimensional structure to the Vh domain of the murine antibody, and wherein the Vl domain framework regions are derived from a human antibody having a Vl domain that has the most similar three-dimensional structure to the Vl domain of the murine antibody.
  • the humanized antibody comprises Vh domain framework regions derived from IGHV3-21, IGHV3-11, IGHV3-23, IGHV1-69, or IGHV3-48.
  • the humanized antibody comprises Vl domain framework regions are derived from IGKV3-15, IGKV3-20, IGKV1-39, IGKV3-11, or IGKV1-5.
  • CDR grafting is the traditional approach, where non-human CDRs are engineered onto human framework regions, while retaining only those murine framework residues deemed important for the integrity of the antigen-binding site. (Jones et al., Nature, 1986, 321:522-25.)
  • There are several strategies for selecting the human framework In the “fixed frameworks” strategy, the human framework is fixed regardless of the parental antibody or its sequence similarity.
  • human myeloma antibodies REI for the light chain and NEW for the heavy chain are used.
  • human frameworks are selected based on shared sequence similarities to the parental antibody’s variable regions.
  • human frameworks are based on the consensus sequence of subgroups in the Kabat database. The consensus sequence of each Kabat subgroup is composed of the most frequent amino acid at each framework position. Consensus sequences for the Vh and Vl most similar to the non-human sequences are chosen for CDR grafting.
  • human immunoglobulin germline genes most similar to the non-human Vl and Vh sequences are selected.
  • SDR grafting is similar to CDR grafting, but involves grafting a subset of residues in the CDRs that are more variable and are directly involved in the interaction with antigen as compared to more conserved residues in CDRs that maintain the conformations of CDR loops.
  • An SDR-grafted humanized antibody is constructed by grafting the SDRs and the residues maintaining the conformations of the CDRs onto a human template/scaffold/framework.
  • the choice of a human template can be based on selecting human antibody framework sequence(s) that exhibit the closest Vh/Vl angles compared to those in the parental non-human antibody for a correct positioning of the CDRs in the humanized construct.
  • the SDRs are identified from the 3D structure of the antigen-antibody complex, computational analysis of three-dimensional structures of antibody:antigen complexes in databases, and/or by mutational analysis of the antibody-combining site. (De Pascalis et al., J. Immunol., 2002, 169:3076-84.) SDRs are mainly in CDRH1, in the N-terminal and middle regions of CDRH2, CDRH3 but not in the terminal region, C-terminal region of CDRL1, the first and sometimes middle parts of CDRL2, and in the middle region of CDRL3.
  • Reshaping or veneering involves replacing only the surface residues of the non- human variable regions with human residues while maintaining the non-human core and CDRs.
  • Reshaping or veneering involves replacing only the surface residues of the non- human variable regions with human residues while maintaining the non-human core and CDRs.
  • Surface residues can be identified according to a defined set of positions in the heavy and light chain variable regions that are thought to describe the exposed framework surface of the Fv regions, and those residues that are non-human are subsequently backmutated. (Id..; see also Staelens et al., Mol.
  • Superhumanization is also a CDR-grafting approach, but it focuses on structural homologies between the non-human CDRs and human CDRs.
  • Superhumanization involves selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, preferably germline antibody gene segments. Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences.
  • Human germline V genes are identified that have the same canonical structure class as the non-human antibody to be humanized, and those human gene segments are selected whose CDRs have the best residue-to-residue homology to the non-human antibody. In the selected sequences, non-homologous CDR residues in the human gene segments are converted to the non-human antibody sequence.
  • HSC Human string content
  • human frameworks encompass all known heavy and light chain human germline genes. Libraries of the CDR-framework pools are cloned into phage expression vectors and screened for binding to the antigen. Framework shuffling does not require rational design from sequence analysis, structural information, or backmutations. [0224] In “human framework adaptation (HFA),” human germline genes are selected based on sequence and structural considerations. (Fransson et al.2010.) An expression library is first constructed by combining the binding site of the non-human antibody with human germline genes. The binding site includes CDRs (Kabat definition) and also hypervariable loops (Chothia definition).
  • Antibodies preferably monoclonal antibodies, according to the invention can be made by any method known in the art.
  • plasma cells are cultured in limited numbers, or as single plasma cells in microwell culture plates.
  • Antibodies can be isolated from the plasma cell cultures. Vh and Vl can be isolated from single cell sorted plasma cells. From the plasma cell, RNA can be extracted, and PCR can be performed using methods known in the art.
  • Vh and Vl regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into intermediate vectors for further engineering or into an expression vector that is then transfected into HEK293T cells or other host cells as described below or known in the art.
  • RT-PCR reverse transcriptase PCR
  • the cloning of nucleic acid in intermediate vectors, expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.
  • Antibody isolation and purification techniques are known in the art, which can include filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • the antibodies or fragments of the invention have an IgM Fc region or constant domains thereof. It is established that IgM can assume both pentameric and hexameric configurations, depending on the substitution of the J-chain with an additional Fab(2) monomer, which increases the number of Fabs on a single IgM from 10 to 12 (Hiramoto et al Sci.
  • Stable or transient IgM producing cells lines can be generated as described in Chromikova et al., 2015 and Hennicke et al., 2020, where two different pIRES expression vectors are used, one to express the heavy chain and the other to express the light chain and the J-chain sequence.
  • IgM antibodies can be purified according to standard methods in the art, including IgM specific resins for use in affinity chromatography (e.g., POROS Capture Select IgM Affinity Matrix by ThermoFisherScientific.) Transmission electron microscopy (TEM) can be used to confirm pentameric and hexameric forms of IgM. [0228] In addition.
  • protein fragments of antibodies can be obtained by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof.
  • Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab')2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs.
  • Eukaryotic, e.g., mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules.
  • Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Mammalian cell lines suitable for expression of therapeutic antibodies are well known in the art.
  • the antibody molecule may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells.
  • the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide.
  • a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.
  • antibodies according to the invention may be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, e.g. by use of a vector according to the present invention, and (ii) isolating the expressed antibody product.
  • the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of the desired specificity or function.
  • the screening step may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function.
  • the assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their differentiation status, etc.
  • Individual transformed B cell clones may then be produced from the positive transformed B cell culture.
  • the cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art.
  • Nucleic acid from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.
  • B cell clones or transfected host-cells of the invention can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.
  • Expression from recombinant sources is common for pharmaceutical purposes than expression from B cells or hybridomas e.g., for reasons of stability, reproducibility, culture ease, etc.
  • the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and/or light chain mRNAs) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a host cell in order to permit expression of the antibody of interest in that host cell.
  • nucleic acids e.g., heavy and/or light chain mRNAs
  • the invention provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing nucleic acid(s) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; and (ii) using the sequence information from step (i) to prepare nucleic acid(s) for insertion into a host cell in order to permit expression of the antibody of interest in that host cell.
  • the nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, to change codon usage, and/or to optimize transcription and/or translation regulatory sequences.
  • the invention also provides a method of preparing a transfected host cell, comprising the step of transfecting a host cell with one or more nucleic acids that encode an antibody of interest, wherein the nucleic acids are nucleic acids that were derived from a cell sorted B cell or a cultured plasma cell of the invention.
  • nucleic acids are nucleic acids that were derived from a cell sorted B cell or a cultured plasma cell of the invention.
  • These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production. They can also be used as the active ingredient of a pharmaceutical composition.
  • Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites fluid, hollow-fiber type bioreactor cartridge, modular minifermenter, stirred tank, microcarrier culture, ceramic core perfusion, etc.
  • Any suitable host cells could be used for transfection and production of the antibodies of the invention.
  • the transfected host cell may be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a human liver cell), as well as plant cells.
  • expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans.
  • the transfected host cell may be able to grow in serum-free media. In a further embodiment, the transfected host cell may be able to grow in culture without the presence of animal-derived products. The transfected host cell may also be cultured to give a cell line.
  • protein therapeutics are produced from mammalian cells. The most widely used host mammalian cells are Chinese hamster ovary (CHO) cells and mouse myeloma cells, including NS0 and Sp2/0 cells. Two derivatives of the CHO cell line, CHO-K1 and CHO pro-3, gave rise to the two most commonly used cell lines in large scale production, DUKX-X11 and DG44.
  • mammalian cell lines for recombinant antibody expression include, but are not limited to, COS, HeLa, HEK293T, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, HEK 293, MCF-7, Y79, SO-Rb50, HepG2, J558L, and BHK. If the aim is large-scale production, the most currently used cells for this application are CHO cells. Guidelines to cell engineering for mAbs production were also reported.
  • the invention provides an antibody, or antibody fragment, that is recombinantly produced from a mammalian cell-line, including a CHO cell-line.
  • the invention provides a composition comprising an antibody, or antibody fragment, wherein the antibody or antibody fragment was recombinantly produced in a mammalian cell-line, and wherein the antibody or antibody fragment is present in the composition at a concentration of at least 1, 10, 100, 1000 micrograms/mL, or at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 milligrams/mL.
  • a concentration of at least 1, 10, 100, 1000 micrograms/mL or at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 milligrams/mL.
  • the antibody composition comprises less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 50, or 100 nanograms of host cell protein (i.e., proteins from the cell-line used to recombinantly produce the antibody)). In other embodiments, the antibody composition comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 ng of protein A per milligram of antibody or antibody fragment (i.e., protein A is a standard approach for purifying antibodies from recombinant cell culture, but steps should be done to limit the amount of protein A in the composition, as it may be immunogenic). (See, e.g., U.S.
  • nucleic acids encoding the inventive antibodies.
  • the nucleic acids are mRNA, modified or unmodified, suitable for use any use, e.g. but not limited to use as pharmaceutical compositions.
  • the nucleic acids are formulated in lipid, such as but not limited to LNPs.
  • the invention provides a method for making recombinant HLA-E-VL9 specific antibodies by screening for very rare antibodies from a circulating B- cell antibody repertoire.
  • the method has a sensitivity level where it can identify and isolate B-cells expressing HLA-E-VL9 specific antibodies that are present at a very low percentage as compared to the overall circulating B-cell population, i.e., 1 in 1 million, 1 in 2 million, 1 in 3 million, 1 in 4 million, 1 in 5 million, 1 in 10 million cells, or 1 in 100 million cells or more.
  • the method comprises the following steps: (1) Fold a VL9 peptide (or other test peptide) with HLA-E to make a stable complex; (2) Assemble the folded HLA-E-peptide as a tetramer; (3) Use the tetramer to stain B cells from peripheral blood of a human donor or an animal (the donor or animal may be pre-immunized or challenged; for example, mice can be immunized with HLA-E-peptide, or for example, if one is preparing an antibody to an HLA-E-pathogen peptide complex, then one can screen human donors infected or immunized with the pathogen or animals immunized with the HLA-E- pathogen peptide complex); (3) Sort tetramer binding B cells as single cells and clone DNA or mRNA for antibody heavy and light chains; (4) Express full length DNA for heavy and light chains in suitable cells (e.g., HEK293T) so that antibody is expressed and
  • standard approaches can be used for staining and sorting for B-cells that express antibodies specific to an HLA-E-peptide complex.
  • FACS fluorescent activated cells sorting
  • numerous commercial reagents are available to stain cells with antibodies conjugated to different fluorescent colors, including different combinations of reagents to identify and sort B-cells and B-cell sub- populations such as na ⁇ ve and memory B-cells.
  • the tetramers are conjugated to fluorescent dyes according to standard methods in the art.
  • the present invention also provides a pharmaceutical composition comprising one or more of: (i) the antibody, or the antibody fragment thereof, according to the present invention; (ii) the nucleic acid encoding the antibody, or antibody fragments according to the present invention; (iii) the vector comprising the nucleic acid according to the present invention; and/or (iv) the cell expressing the antibody according to the present invention or comprising the vector according to the present invention.
  • the invention provides a pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention and/or the cell according to the present invention.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier, diluent and/or excipient.
  • the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • pharmaceutically acceptable carriers in a pharmaceutical composition according to the present invention may be active components or inactive components. In certain embodiments the pharmaceutically acceptable carrier in a pharmaceutical composition according to the present invention is not an active component in respect to coronavirus infection.
  • Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
  • Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol.
  • compositions of the invention may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis.TM. and Herceptin.TM., for reconstitution with sterile water containing a preservative).
  • the composition may be prepared for topical administration e.g., as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored).
  • the composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g., as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject.
  • a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.
  • a thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.
  • Pharmaceutical compositions of the invention generally have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, and in other embodiments about 7. The pH may be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen free.
  • the composition may be isotonic with respect to humans.
  • pharmaceutical compositions of the invention are supplied in hermetically-sealed containers.
  • compositions present in several forms of administration include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion.
  • parenteral administration e.g., by injection or infusion
  • the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents.
  • the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid.
  • a vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention.
  • the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present invention.
  • compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions.
  • the pharmaceutical composition is an injectable.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also contemplated, e.g. that the pharmaceutical composition is in lyophilized form.
  • the active ingredient could be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Whether it is a polypeptide, peptide, or nucleic acid molecule, other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is in a "prophylactically effective amount” or a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.
  • the pharmaceutical composition according to the present invention may be provided for example in a pre-filled syringe.
  • inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • the active ingredient i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers.
  • Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the inventive pharmaceutical composition can be formulated in a suitable lotion or cream.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Suitable dose ranges can depend on the antibody (or fragment) and on the nature of the formulation and route of administration. For example, doses of antibodies in the range of 0.1-50 mg/kg, 1-50 mg/kg, 1-10 mg/kg, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/kg of antibody can be used.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the pharmaceutical composition may be provided as single-dose product.
  • the amount of the antibody in the pharmaceutical composition--in particular if provided as single-dose product--does not exceed 200 mg. In certain embodiments, the amount does not exceed 100 mg, and in certain embodiments, the amount does not exceed 50 mg.
  • the antibodies of the invention could be used for non therapeutic uses, such as but not limited to diagnostic assays.
  • the antibodies are administered as nucleic acids, including but not limited to mRNAs which could be modified and/or unmodified.
  • the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector.
  • the compositions comprise a suitable carrier.
  • the compositions comprise a suitable adjuvant.
  • the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter.
  • the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter.
  • the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro.
  • the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention.
  • the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention.
  • the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention.
  • the nucleic acid of the invention is operably linked to a promoter and is inserted in an expression vector.
  • the invention provides a composition comprising the expression vector. [0266]
  • the invention provides a composition comprising at least one of the nucleic acid sequences of the invention.
  • the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.
  • the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the inventive sequences. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of the sequences in in the instant application, or a variant thereof or a fragment thereof.
  • the invention provides an RNA molecule encoding one or more of inventive antibodies.
  • the RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.
  • a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp). This cap can enhance in vivo translation of the RNA.
  • the 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group.
  • a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge.
  • a RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end.
  • a RNA molecule useful with the invention may be single-stranded.
  • a RNA molecule useful with the invention may comprise synthetic RNA.
  • the recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation.
  • optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).
  • a kozak sequence e.g., GCC ACC
  • Ig immunoglobulin leader sequence encoding a signal peptide
  • Ig immunoglobulin leader sequence encoding a signal peptide
  • eliminating to the extent possible cis-acting sequence motifs i.e., internal TATA boxes.
  • the methods lead to protection or treatment of infection or disease by blocking or otherwise inhibiting the intercellular signaling mediated by the engagement of an HLA-E-VL9 complex (or an HLA- E peptide of interest complex such as a tumor or viral peptide) on an infected or diseased cell and the NKG2A receptor expressed on sub-populations of NK cells and CD8+ T-cells.
  • an HLA-E-VL9 complex or an HLA- E peptide of interest complex such as a tumor or viral peptide
  • Tumor cells and virally-infected cells have been known to co-opt this intercellular signaling pathway in order to inhibit the activation and concomitant killing by the NKG2A+ NK cell and NKG2A+ CD8+ T-cell sub-populations.
  • activated NKG2A+ NK cell and NKG2A+ T-cells include such cells that downregulate and reduce or eliminate cell- surface expression of NKG2A+.
  • the therapeutic method is for protection against cytomegalovirus HCMV, as an HLA-E-HCMV peptide complex has a peptide with an exact match to one of the VL9 peptide sequences VMAPRTLIL that is expressed and binds to HLA-E.
  • the therapeutic method comprises administration of an antibody having the binding specificity of the 3H4 antibody, is a chimeric version of 3H4, or is a humanized version of 3H4.
  • the therapeutic compositions and methods not only involve blocking the inhibitory HLA-E-VL9-NKG2A pathway in said NK cells and CD8+ T- cells, but also: (1) blocking other inhibitory receptors on these NK cells and CD8+ T-cells, and/or (2) promoting the activation of stimulatory receptors on these NK cells and CD8+ T- cells.
  • the multiple targeting of receptors on NKG2A+ NK cell and NKG2A+ CD8+ T-cell sub-populations can be accomplished, for example, by the use of combination of different antibodies or agents each targeting a different receptor, or by recombinant multi-specific antibodies.
  • the invention provides methods of treatment comprising administering the pharmaceutical composition of the invention.
  • the methods are applicable to diseases or conditions that would benefit from an increase in the number of stimulated effector immune cells such as NK cells, CD8+ T-cells, and JG T-cells (which can also mediate cytotoxic responses against tumors).
  • exemplary diseases or conditions include, but are not limited to, cancer and viral infection.
  • Such methods of treatment can relate to methods of immunostimulation comprising the steps of: administering to a subject in need of enhanced immune cytotoxic effector function a therapeutically effective amount of an antibody of the invention, which antibody specifically binds to at least an HLA-E-VL9 peptide complex (or other HLA-E-peptide of interest complexes) and increases the number of activated NK cells or activated CD8+ cells.
  • An increase in the number of activated NK cells or activated CD8+ cells can be determined by testing for the ability of the antibody to increase the proportion of activated cells in a sample, such as a peripheral blood sample.
  • activated NK cells can be identified by a decrease in CD16 surface level and an increase in CD107a. (See, e.g., Veluchamy et al., Scientific Reports, 2017, 7:43873.)
  • activated effector T-cells can be identified by a loss of L-selectin, a gain of VLA-4, higher levels of LFA-1 and CD2, and expression of CD45RO instead of CD45RA.
  • the 51 Cr release assay provides an in vitro means to determine whether an antibody of the invention can cause an increase in NK cell activation.
  • the methods comprise administering additional therapeutic or prophylactic agents, including but not limited to additional antibodies that block inhibitory receptors on NK or T-cells, antibodies that work as agonists for stimulatory receptors on NK or T cells, small molecule therapeutics, or any other suitable agent.
  • the additional agent is an antibody that specifically binds to the NKG2A/CD94 complex on NK cells or CD8+ T-cells.
  • the additional agent is an antibody that specifically binds the CTLA-4 receptor on NK cells or CD8+ T-cells.
  • the additional agent is an antibody that specifically binds PD-1 on NK cells or CD8+ T-cells.
  • the methods comprise administering one or more antibodies or antigen fragments thereof, including without limitation multimeric antibodies, of the invention in a combination treatment. In certain embodiments, the these are selected such that each antibody or antigen fragments thereof has at least one differential function compared to other antibody or antibodies in the combination. In a non-limiting embodiment, antibodies or antigen binding fragments in a combination treatment have non-overlapping epitopes. In non-limiting embodiments, antibodies or antigen fragments thereof in a combination treatment provide different effect on cellular pathways. [0277] In some embodiments, a combination treatment comprising inventive antibodies could further comprise additional therapeutic or prophylactic agents. [0278] Table 4. Non limiting embodiments of VL-9 peptides
  • a dash in the table indicates that the amino acid at that position is identical to the amino acid in the VMAPRTLLL peptide.
  • References are as follows: (1) www.ebi.ac.uk/ipd/imgt/hla/; (2) See Example 1, (3) Walters LC, Harlos K, Brackenridge S, Rozbesky D, Barrett JR, Jain V, Walter TS, O'Callaghan CA, Borrow P, Toebes M, Hansen SG, Sacha JB, Abdulhaqq S, Greene JM, Fruh K, Marshall E, Picker LJ, Jones EY, McMichael AJ, Gillespie GM.
  • HLA-E human leukocyte antigen E
  • VL9 HLA E VL9 complexes interact with the natural killer (NK) cell receptors NKG2A C/CD94 and regulate NK cell-mediated cytotoxicity.
  • 3H4 a murine HLA-E-VL9-specific IgM antibody that enhanced killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line.
  • Structural analysis revealed that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9.
  • an affinity-optimized IgG form of 3H4 showed enhanced NK killing of HLA-E-VL9-expressing cells.
  • HLA-E-VL9-specific IgM antibodies similar in function to 3H4 were also isolated from na ⁇ ve B cells of cytomegalovirus (CMV)- negative, healthy humans.
  • CMV cytomegalovirus
  • NK cells play critical roles in immune surveillance by discriminating normal from altered cells, and function as effector cells by killing non-self malignant or pathogen-infected cells and by producing inflammatory cytokines (Chiossone et al., 2018; Raulet, 2006; Yokoyama and Kim, 2006).
  • NK cell inhibitory receptors ligate human lymphocyte antigen (HLA) or major histocompatibility complex (MHC) class I molecules expressed on healthy cells as self. Conversely, cells lacking MHC class I are recognized by NK cells as “missing-self” and are sensitive to NK cell-mediated killing (Ljunggren and Karre, 1985, 1990).
  • HLA human lymphocyte antigen
  • MHC major histocompatibility complex
  • KIRs recognize classical human HLA class Ia molecules (Colonna and Samaridis, 1995; Karlhofer et al., 1992; Pende et al., 2019), whereas the inhibitory NKG2A/CD94 heterodimeric receptor interacts with the non-classical HLA class Ib molecule HLA-E and is balanced by an activating receptor NKG2C/CD94 (Braud et al., 1997; Braud et al., 1998; Brooks et al., 1997).
  • HLA-E has limited polymorphism with only two expressed variants, HLA-E*01:01 and HLA-E*01:03, that differ only in residue 107, which is outside the peptide-binding groove (Kraemer et al., 2014).
  • the NKG2A/CD94/HLA-E pathway is considered as an important immune checkpoint target and has recently become a focus for NK cell-based immunotherapeutic strategies (Andre et al., 2018; Hu et al., 2019; Kim et al., 2019; Souza-Fonseca-Guimaraes et al., 2019; van Hall et al., 2019).
  • a subset of CD8+ T cells also express NKG2A/CD94, and inhibition of NKG2A/CD94 - HLA-E interaction similarly has application in CD8+ T cell-based immunotherapy (Andre et al., 2018; van Montfoort et al., 2018).
  • HLA-E engages with NKG2A/CD94 via a restricted subset of peptides VMAPRT(L/V) (V/L/I/F)L (designated VL9) that derive from the leader sequence of HLA- A, -C, -G and a third of HLA-B molecules (Braud et al., 1997; Braud et al., 1998; Lee et al., 1998a; Lee et al., 1998b).
  • HLA-E binds VL9 peptides, which stabilize HLA-E surface expression (Braud et al., 1997; Braud et al., 1998) on healthy host cells in which HLA-Ia expression is not perturbed and initiate recognition by NKG2A/CD94 or NKG2C/CD94 on NK cells.
  • the binding affinity of HLA-E-VL9 peptide complexes for NKG2A/CD94 is greater than that for NKG2C/CD94, such that the inhibitory signal dominates to suppress aberrant NK cell-mediated cytotoxicity and cytokine production (Aldrich et al., 1994; Braud et al., 1998; Kaiser et al., 2008; Llano et al., 1998; Rolle et al., 2018).
  • HLA-E and its murine or rhesus macaque homologs are capable of binding to a range of other host peptides and pathogen-derived peptides, including heat-shock protein 60 (Hsp60)-derived peptides (Michaelsson et al., 2002), Mycobacterium tuberculosis (Mtb) peptides (Joosten et al., 2010; van Meijgaarden et al., 2015), and simian immunodeficiency virus (SIV) Gag peptides (Hansen et al., 2016; Walters et al., 2018).
  • Hsp60 heat-shock protein 60
  • Mtb Mycobacterium tuberculosis
  • SIV simian immunodeficiency virus
  • leader sequence VL9 peptides are essential not only for stabilizing HLA-E surface expression but also for mediating the role of HLA-E/NKG2A/CD94 in regulating NK cell self-recognition.
  • interruption of this pathway by specifically targeting HLA-E-peptide complexes on target cells can enhance NK cell activity.
  • Natural antibodies are immunoglobulins that are present prior to simulation by cognate antigen, and provide the first line of defense against bacterial, fungal and viral infections (Holodick et al., 2017). They also suppress autoimmune, inflammatory and allergic responses, protect from atherosclerotic vascular injury, and mediate apoptotic cell clearance (New et al., 2016). Natural antibodies are generally near germline in sequence, have repertoire skewing of variable heavy chain (VH) and variable light chain (VL) genes, and respond to antigens with T cell independence (Holodick et al., 2017). However, specific roles of natural antibodies in regulation of natural killer (NK) cell function are unknown.
  • VH variable heavy chain
  • VL variable light chain
  • 3H4 mAb enhanced NK cytotoxicity as an IgM
  • the IgG form of the antibody showed no such functionality.
  • 3H4 IgG variants with enhanced HLA-E-VL9 binding by highthroughput screening of antibody libraries.
  • These optimized 3H4 IgG Abs contain mutations in their CDR-H3 loops, bind HLA-E/VL-9 ⁇ 220 times tighter than the WT mAb and showed robust enhancement of NK cytotoxicity.
  • human HLA-E-VL9-reactive, near-germline IgMs were isolated from the human na ⁇ ve B cell repertoire that also enhanced NK cell killing as IgG.
  • HLA-E-VL9-specific mAb 3H4 is a minimally mutated pentameric IgM
  • Sequence analysis of 3H4 mAb revealed 1.04% heavy chain variable region (VH) and 2.51% light chain viable region (VL) mutations (Table 5).
  • 3H4 IgM recognized the ⁇ 1/ ⁇ 2 domain of HLA-E and N-terminus of the VL9 peptide
  • 3H4 binding to VL9 peptide presented by HLA-E we tested 3H4 binding to VL9 peptide presented by HLA-E, the rhesus ortholog Mamu-E, as well as two HLA-E/Mamu-E hybrids – one with HLA-E ⁇ 1/Mamu-E ⁇ 2 (H ⁇ 1/M ⁇ 2), the other with Mamu-E ⁇ 1/HLA-E ⁇ 2 (M ⁇ 1/H ⁇ 2).
  • 3H4 did not bind to Mamu-E/VL9 or H ⁇ 1/M ⁇ 2-VL9, and its staining of cells expressing M ⁇ 1/H ⁇ 2-VL9 was weak (Figure 1E), suggesting that 3H4 recognition involved interaction with both ⁇ 1 and ⁇ 2 domains of HLA-E, and the epitope on ⁇ 2 might be partially conserve
  • VL9 mutations indicated that position 1 (P1) of the peptide was important for 3H4 binding ( Figure 1F), with strong antibody recognition of VL9 peptide P1 variants with alanine, cysteine, isoleucine, serine, threonine, weak binding to histidine and proline substitutions, but no interaction with arginine, glutamate, glycine, lysine, methionine, asparagine, tryptophan, tyrosine or phenylalanine ( Figures 1G and 12K). These data suggested that mAb 3H4 made contacts with both the HLA-E ⁇ 1/ ⁇ 2 domain and the amino-terminal end of the VL9 peptide.
  • the 3H4-HLA-E interface was mainly mediated via electrostatic interactions and was dominated by the 3H4 VH chain segment which created a total buried surface area of 1109.4 ⁇ 2 and formed ten hydrogen bonds (H-bonds) and three salt bridges with HLA-E residues of the ⁇ 1-helix and one H-bond with T163 of the HLA-E ⁇ 2-helix.
  • the smaller 3H4 VL chain-HLA-E interface buried 522.8 ⁇ 2 and involved only three inter-molecular H-bonds and three salt bridges ( Figures 15-17).
  • the VH CDR2 region (residues I51-T57) was positioned above the HLA-E ⁇ 1-helix where a string of inter- molecular H-bonds were formed involving G56 and N54 of the VH CDR2 in addition to D50, Q61 and K64 of the framework VH chain region (Figure 2H).
  • R65 of the HLA-E ⁇ 1-helix formed four H-bonds with the 3H4 VH and also mediated polar pi stacking interactions with W100D of the VH CDR3 loop.
  • the optimized 3H4 mAbs enhanced NK-92 cell killing of HLA-E-VL9-transfected 293T cells at concentrations of 10 ⁇ g/ml and 1 ⁇ g/ml to levels comparable to those observed for 3H4 IgM ( Figures 4G and 18D). Therefore, the higher affinity of affinity-optimized 3H4 IgG for HLA-E-VL9 could compensate for the need for avidity effect in the 3H4 IgM multimers to mediate NK enhancement.
  • HLA-E-VL9-specific antibodies exist in CMV-negative, healthy humans [0307] That HLA-E-VL9-specific antibodies were isolated from mice immunized with an unrelated peptide antigen (RL9HIV) implied that antibody 3H4 might be derived from the natural B cell pool rather than induced by immunization. Therefore, we assessed binding of HLA-E-VL9 fluorescent tetramers to B220+CD19+ B cells from na ⁇ ve HLA-B27/ ⁇ 2M TG mice and B6 mice and found that HLA-E-VL9-tetramer-binding B cells existed in unimmunized mice ( Figures 20A-B).
  • HLA-E-VL9-specific mouse antibodies were minimally mutated IgM antibodies (Table 5). These findings raised the hypothesis that HLA-E-VL9-specific antibodies were natural antibodies in mice. [0308]
  • HLA-E-VL9 tetramers as probes, we identified B cells expressing HLA-E-VL9-specific B cell receptors (BCRs) in four male, cytomegalovirus (CMV) seronegative human donors ( Figures 5A and 18C, Table 6).
  • HLA-E-VL9-specific B cells were IgD+IgM+/- B cells, in which four cell subsets were observed ( Figure 5E) – CD10-CD27-CD38+/- na ⁇ ve B cells (71.4%), CD10+CD27-CD38++ immature or newly formed B cells (Giltiay et al., 2019) (10.7%), and CD10-CD27+CD38- non-class-switched memory cells, demonstrating that BCRs specifically targeting HLA-E- VL9 peptide existed in the na ⁇ ve B cell repertoire of healthy humans.
  • H /V L gene usage of HLA-E-VL9-specific antibodies [0311] Natural antibodies demonstrate Ig repertoire skewing (Holodick et al., 2017; New et al., 2016). To characterize the human antibody gene usage of HLA-E-VL9 antibodies, we analyzed the paired heavy chain and light chain gene sequences of 56 human HLA-E-VL9 antibodies and found 1 multiple-member clone containing 6 antibodies in donor LP021 (Kepler et al., 2014) ( Figure 22). Next, we compared the 51 HLA-E-VL9-specific B cell clones with a reference human antibody repertoire (DeKosky et al., 2015).
  • HLA-E-VL9 antibodies showed a trend to have shorter heavy chain complementarity determining region 3 (CDR3) lengths than reference antibodies (Figure 5H), while no difference was observed for light chain CDR3 ( Figure 5I).
  • CDR3 complementarity determining region 3
  • HLA-E-VL9-specific antibodies were IgM, minimally mutated and displayed skewed usage of VH and V ⁇ /V ⁇ genes.
  • HLA-E-VL9-specific mAbs recognize microbiome-derived VL9-like peptides presented by HLA-E
  • HLA-E binding scores Eight peptides with the highest HLA-E binding scores were synthesized as 9 amino acid peptides, incubated with K562-E cells, and tested for mAb 3H4 HLA-E-VL9-specific antibody binding. Seven out of eight microbiome sequence- derived peptides showed strong HLA-E binding as indicated by the ability to stabilize and upregulate HLA-E expression, as determined by detecting with the HLA-E reactive antibody, 3D12 ( Figures 5L and 21E).
  • VMAPRTLLL VL9
  • VMPPRALLL from Escherichia coli MS 175-1
  • VMAGRTLLL from Stenotrophomonas sp.
  • VMAPRTKLL from Pseudomonas formosensis
  • microbiome-derived peptides in complex with HLA-E were capable of binding to HLA-E-VL9-specific antibodies and raised the hypothesis that microbiome peptides may be one type of antigen capable of stimulating B cells with HLA-E-VL9 peptide specificity in vivo.
  • DISCUSSION [0315] In this study, we have isolated and characterized antibodies reactive with HLA-E- VL9 peptide complexes, and found these antibodies were derived from the na ⁇ ve IgM B cell BCR repertoire in mice as well as in non-immunized, HCMV seronegative male humans.
  • HLA-E-VL9 Somatic mutations of these antibodies were minimal, and the affinities of these antibodies for HLA-E-VL9 were low.
  • the lack of class-switching in HLA-E-VL9-specific antibodies may reflect self-tolerance of CD4 T cells and a lack of T cell help for affinity maturation of these antibodies.
  • the mouse antibodies were selected in the setting of HLA-E-unrelated peptide immunizations, they were minimally mutated IgM antibodies, as were the antibodies isolated from human CMV-negative, healthy males.
  • HLA-E-VL9- 3H4 Fab co-complex revealed that the 3H4 heavy chain made key contacts with HLA-E and the VL9 peptide using germline-encoded residues in the CDR-H3 (D) region.
  • 3H4 is a mouse antibody that reacted with human HLA-E-VL9.
  • the HLA-E equivalent in C57BL/6xSJL mice is Qa1b which presents a similar class Ia signal peptide AMAPRTLLL and 3H4 did not bind to this HLA-E-peptide complex.
  • HLA-E-VL9-specific antibodies were identified in the na ⁇ ve B cell pool of healthy humans and, like the mouse 3H4, the human CA147 HLA-E-VL9 antibody enhanced NK cytotoxicity of NKG2A+ NK cells. Therefore, this type of natural antibody-producing B cell could play immunoregulatory roles in humans to enhance NK killing of pathogen infected cells in the early stages of a viral infection. If so, this might provide the selective force to maintain these enriched V genes in the germline.
  • HLA-E antibodies in non- alloimmunized humans could be elicited by autoantigens derived from viral, bacterial, or environmental agents cross-reactive with HLAs, or soluble HLA-E heavy chains that become immunogenic without the ⁇ 2M subunit (Alberu et al., 2007; Hickey et al., 2016; Ravindranath et al., 2010a; Ravindranath et al., 2010b).
  • a recent study found that mouse gut microbial antigens shaped the BCR repertoire by contributing to BCR selection and affinity maturation (Chen et al., 2020).
  • HLA-E binding peptides with sequence similarities to VL9, derived from the human microbiome.
  • Three of our HLA-E-VL9 antibodies recognized a subset of these HLA-E-presented microbiome-derived VL9-like peptides.
  • human microbial peptides presented by HLA-E could potentially interact with HLA-E-VL9-bound na ⁇ ve BCRs, and trigger expansion of B cells that express HLA-E-VL9-specific BCRs.
  • Viruses, bacteria and other microbes stimulate such innate adaptive immune interactions.
  • CMV human cytomegalovirus
  • VMAPRTLIL VL9 sequence VMAPRTLIL in the leader sequence of its UL40 gene.
  • This peptide is processed in a TAP independent manner and presented bound to HLA-E at the cell surface to inhibit NK cell killing and evade innate immune responses (Tomasec et al., 2000). This has not been reported to elicit antibody responses, but HLA-E-UL40 peptide-specific T cells have been described when the limited polymorphism in the HLA A, B and C sequences mismatches that of the virally-encoded VL9 peptide sufficiently to overcome self-tolerance (Sullivan et al., 2015).
  • Monalizumab the first-in-class monoclonal antibody checkpoint inhibitor targeting NKG2A, enhances anti-tumor immunity by activating cytotoxic activities of effector CD8+ T cells and NK cells (Andre et al., 2018; Creelan and Antonia, 2019; van Hall et al., 2019).
  • NK inhibitory receptor NKG2A/CD94 co-complex structural analysis revealed steric clashes between the 3H4 Fab and the NK inhibitory receptor NKG2A/CD94 when docked onto HLA-E-VL9, which explained the mechanism of 3H4 IgM enhancing NKG2A+ NK cell killing.
  • mouse 3H4 IgM, the affinity-optimized 3H4 IgG, and the recombinant IgG1 form of human CA147 both enhanced the cytotoxicity of an NKG2A+ human NK cell line NK92, which is a safe and established cell line for adoptive immunotherapy in phase I clinical trials (Klingemann et al., 2016).
  • HLA-E-VL9- targeting antibodies 3H4 and CA147 could have therapeutic potential as NK checkpoint inhibitors.
  • our study has demonstrated a novel specificity of IgM natural antibodies, that of recognition of HLA-E-VL9 peptide complexes, which suggests an NK cell immunoregulatory role by a subset of natural antibodies.
  • HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795-799.
  • NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor. J Exp Med 185, 795-800.
  • HLA-E Presentation of a Broader Peptide Repertoire Impacts the Cellular Immune Response-Implications on HSCT Outcome. Stem Cells Int 2015, 346714.
  • Alternative peptide repertoire of HLA-E reveals a binding motif that is strikingly similar to HLA-A2.
  • HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160, 4951-4960.
  • HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A.
  • KIRs Killer Ig-Like Receptors
  • HLA-E monoclonal antibodies recognize shared peptide sequences on classical HLA class Ia: relevance to human natural HLA antibodies. Mol Immunol 47, 1121-1131. [0372] Rolle, A., Meyer, M., Calderazzo, S., Jager, D., and Momburg, F. (2016). Distinct HLA-E Peptide Complexes Modify Antibody-Driven Effector Functions of Adaptive NK Cells. Cell Rep 24, 1967-1976 e1964. [0373] Sathe, A., and Cusick, J.K. (2021). Biochemistry, Immunoglobulin M. In StatPearls (Treasure Island (FL)).
  • HLA-B27 transgenic mice as potential models of human disease. In Transgenic mice and mutants in MHC research (Springer), pp.268-275.
  • HLA-E an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287, 1031.
  • Monalizumab inhibiting the novel immune checkpoint NKG2A. J Immunother Cancer 7, 263.
  • ATCC.293T cells ATCC CRL-3216
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin/streptomycin Gibco, Catalog# 10378016
  • K562 cells ATCC CCL-243
  • K562-E cells and K562-E/UL49.5 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Hyclone, Catalog# SH30228.01) supplemented with 10% FBS.
  • IMDM Iscove's Modified Dulbecco's Medium
  • Jurkat, DU-4475 and U-937 cells were cultured in RPMI-1640 medium (Gibco, Catalog# 72400) supplemented with 10% FBS.
  • SiHa cells were cultured in Minimum Essential Medium (MEM; Gibco, Catalog# 11095080) supplemented with 10% FBS.
  • the NK-92 human cell line (ATCC CRL-2407) was cultured in Alpha Minimum Essential medium ( ⁇ - MEM; Gibco, Catalog# 12561072) supplemented with 2 mM L-glutamine, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100 U/ml recombinant IL-2 (Biolegend, Catalog# 589108), 12.5% horse serum (Gibco, Catalog# 16050122) and 12.5% FBS. All the cells were maintained at 37°C, 5% CO2 in humidified incubators.
  • Alpha Minimum Essential medium ⁇ - MEM; Gibco, Catalog# 12561072
  • 2 mM L-glutamine 2 mM L-glutamine
  • 0.2 mM inositol 0.1 mM 2-mercaptoethanol
  • 0.02 mM folic acid 100 U/ml recombinant IL-2 (Biolegend, Catalog# 589108)
  • mice carrying human ⁇ 2-microglobulin ( ⁇ 2m) and HLA-B*27:05 genes were obtained from Jackson lab (B6.Cg-Tg(B2M,HLA-B*27:05)56-3Trg/DcrJ; stock# 003428). Hemizygous mice were used in this experiment, as this strain is homozygous lethal.
  • peripheral blood lymphocytes PBLs were isolated and stained using mouse CD45 antibody (Biolegend, Catalog# 103122), human HLA class I antibody (Biolegend, Catalog# 311406) and human ⁇ 2m antibody (Biolegend, Catalog# 316312).
  • VMAPRTVLL VL9 peptide
  • Fmoc 9- fluorenylmethoxy carbonyl
  • HLA-E-peptide protein refolding and purification [0395] ⁇ 2-microglobulin, previously purified from inclusion bodies in a Urea-MES buffer, was added to a refolding buffer to achieve a final concentration of 2 ⁇ M.
  • the refold buffer comprised 100 mM Tris pH8.0, 400 mM L-arginine monohydrochloride, 2 mM EDTA, 5 mM reduced glutathione and 0.5 mM oxidized Glutathione and was prepared in MiliQ water.
  • HLA-E-peptide biotinylation and tetramer generation [0396] HLA-E-peptide samples requiring biotinylation were subsequently buffered exchanged on Sephadex G-25 PD10 columns (GE Healthcare, UK) into 10mM Tris buffer using commercially available BirA enzyme (Avidity, USA) following the manufacturer’s instructions. Following overnight biotinylation, protein samples were subsequently purified into 20mM Tris pH8,100mM NaCl buffer or PBS on a HiLoad 16/600 Superdex 75pg column using an AKTA size exclusion fast protein liquid chromatography (FPLC) system.
  • FPLC AKTA size exclusion fast protein liquid chromatography
  • HLA-E*01:03 tetramers were generated via conjugation to various fluorescent labels including Extravidin-PE (Sigma), Streptavidin-bound APC (Biolegend, San Diego) or BV421 (Biolegend, San Diego) at a Molar ratio of 4:1 as previously described (Braud et al., 1998).
  • MAb 3H4 was isolated from this study.
  • SCT single chain trimer
  • MAb 13F11 was isolated from this study.
  • HLA-E-VL9 complex 25 ⁇ g/animal adjuvanted with STR8S-C at Week 0, 2 and 4, following by intraperitoneally (i.p.) immunization with HLA-E-VL9 SCT transfected 293T cells (2x106 cells/animal) at Week 14, 16 and 18.
  • MAb 10C10 and 2D6 were isolated from this study. Serum titers were monitored by ELISA Mice with high binding antibody titers were selected for the subsequent spleen cell fusion and B cell sorting experiments.
  • Hybridoma Cell Line Generation and Monoclonal Antibody Production mice were boosted with the indicated priming antigen 3 days prior to fusion.
  • Spleen cells were harvested and fused with NS0 murine myeloma cells using PEG1500 to generate hybridomas. After 2 weeks, supernatant of hybridoma clones were collected and screened by flow cytometry-based high throughput screening (HTS). Specifically, we tested for antibodies differentially binding 293T cells transiently transfected with plasmid DNA expressing single chain peptide-HLA-E-ß2m trimers so that they expressed HLA-E-RL9HIV, HLA-E-RL9SIV or HLA-E-VL9 at the cell surface.
  • HTS flow cytometry-based high throughput screening
  • Hybridomas cells that secreted HLA-E-VL9 antibodies were cloned by limiting dilution for at least 5 rounds until the phenotypes of all limiting dilution wells are identical.
  • IgG mAbs were purified by protein G affinity chromatography, while IgM mAbs were purified by ammonium sulfate precipitation and by Superose 6 column size-exclusion chromatography in AKTA Fast Protein Liquid Chromatography (FPLC) system.
  • FPLC Fast Protein Liquid Chromatography
  • HLA-E SCT constructs encoding HLA-E-VL9, HLA-E-RL9HIV, or HLA-E- RL9SIV were transfected into 293T cells using GeneJuice transfection reagent (Novagen, Catalog# 70967).
  • GeneJuice transfection reagent Novagen, Catalog# 70967
  • a panel of HLA-E-VL9 SCT constructs with single amino acid mutations were transfected into 293T cells using the same method. Cells were dissociated with 0.1% EDTA at 48 hours post-transfection and stained with a Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, Catalog# L34976).
  • primary antibodies (supernatant from hybridoma cells, supernatant from transfected cells, or purified antibodies) were added and incubated with cells for 1 hour at 4°C, following by staining with 1:1000 diluted secondary antibodies for 30 mins at 4°C.
  • Alexa Fluor 555 conjugated goat anti-mouse IgG (H+L) (Thermo Fisher, Catalog# A32727) or Alexa Fluor 647 (AF647) conjugated goat anti-mouse IgG (H+L) (Thermo Fisher, Catalog# A32728) as secondary antibodies;
  • human primary antibodies we used AF555 conjugated goat anti-human IgG (H+L) (Thermo Fisher, Catalog# A-21433) or AF647 conjugated goat anti-human IgG (H+L) (Thermo Fisher, Catalog# A- 21445) as secondary antibodies.
  • 3H4 Fab production A humanized version of the 3H4 antibody (3H4-huIgG1) was digested to produce Fab fragments using the Pierce Fab Preparation kit (ThermoFisher SCIENTIFIC).3H4 Fab- retrieved sample was further purified by size exclusion on a Superdex S7516/60 column and eluted into PBS buffer.
  • HLA-E-VL9-specific human B cells were sorted in flow cytometry using a three- color sorting technique. Briefly, the stabilized HLA-E- ⁇ 2M-peptide complexes were made as tetramers and conjugated with different fluorophores.
  • Human pan-B cells including na ⁇ ve and memory B cells, were isolated from PBMCs of healthy donors using human pan-B cell enrichment kit (STEMCELL, Catalog# 19554). The isolated pan-B cells were then stained with IgM PerCp-Cy5.5 (Clone# G20-127, BD Biosciences, Catalog# 561285), IgD FITC (Clone# IA6-2, BD Biosciences, Catalog# 555778), CD3 PE-Cy5 (Clone# HIT3a, BD Biosciences, Catalog# 555341), CD235a PE-Cy5 (Clone# GA-R2, BD Biosciences, Catalog# 559944), CD10 PE-CF594 (Clone# HI10A, BD Biosciences, Catalog# 562396), CD27 PE- Cy7 (Clone# O323, eBioscience, Catalog# 25-0279), CD16 BV570 (Clone# 3G8, Biolegend, Catalog# 302035), CD14
  • HLA-E-VL9-specific B cells were sorted in BD FACSAria II flow cytometer (BD Biosciences) for viable CD3neg/ CD14neg /CD16neg /CD235aneg/CD19pos / HLA-E- VL9double-pos/ HLA-E-RL9HIVneg/HLA-E-RL9SIVneg subset as single cells in 96-well plates.
  • VHDHJH and VLJL genes were amplified by RT PCR from the flow cytometry-sorted single B cells using the methods as described previously (Liao et al., 2009; Wrammert et al., 2008) with modification.
  • the PCR-amplified genes were then purified and sequenced with 10 ⁇ M forward and reverse primers. Sequences were analyzed by using the human library in Clonalyst for the VDJ arrangements of the immunoglobulin IGHV, IGKV, and IGLV sequences and mutation frequencies (Kepler et al., 2014).
  • VHDHJH and VLJL sequences were determined as previously described (Liao et al., 2013).
  • Expression of VHDHJH and VLJL as Full-Length IgG Recombinant mAbs [0416] Transient transfection of recombinant mAbs was performed as previously described (Liao et al., 2009). Briefly, purified PCR products were used for overlapping PCR to generate linear human antibody expression cassettes. The expression cassettes were transfected into 293i cells using ExpiFectamine (Thermo Fisher Scientific, Catalog# A14525).
  • the supernatant samples containing recombinant antibodies were used for cell surface staining and HTS assay to measure the binding reactivities.
  • the selected human antibody genes were then synthesized and cloned (GenScript) in a human IgG1 backbone with 4A mutations (Saunders, 2019).
  • Recombinant IgG mAbs were then produced in HEK293i suspension cells by transfection with ExpiFectamine and purified using Protein A resin. The purified mAbs were run in SDS-PAGE for Coomassie blue staining and western blot.
  • Antibodies with aggregation were further purified in AKTA FPLC system using a Superdex 200 size-exclusion column.
  • SPR Surface Plasmon Resonance
  • HLA-E-VL9 complex protein was performed using a BIAcore S200 instrument (Cytiva, formerly GE Healthcare, DHVI BIA Core Facility, Durham, NC) in HBS-EP+ 1x running buffer.
  • the antibodies were first captured onto CM5 sensor chip to a level of ⁇ 9000 RU.
  • the HLA-E-VL9 soluble proteins were injected over the captured antibodies at a flow rate of 30uL/min. After dissociation, the antibodies were regenerated using a 30 second pulse of Glycine pH2.0. Results were analyzed using the Biacore S200 Evaluation software (Cytiva). Subsequent curve fitting analyses were performed using a 1:1 Langmuir model with a local Rmax.
  • ELISA Direct binding ELISAs were conducted in 384-well ELISA plates coated with 2 ⁇ g/ml of C-trap-stabilized HLA-E-VL9, C-trap-stabilized HLA-E-RL9HIV or C-trap- stabilized HLA-E-RL9SIV in 0.1 M sodium bicarbonate overnight at 4°C. Plates were washed with PBS + 0.05% Tween 20 and blocked with 3% BSA in PBS at room temperature for 1 h. MAb samples were incubated for 1 h in 3-fold serial dilutions starting at 100 ⁇ g/ml, followed by washing with PBS-0.05% Tween 20.
  • HRP-conjugated goat anti-human IgG secondary Ab (SouthernBiotech, catalog# 2040-05) was diluted to 1: 10,000 in 1% BSA in PBS-0.05% Tween 20 and incubated at room temperature for 1 h.
  • sandwich ELISA 384- well ELISA plates were coated with HLA-E-VL9 antibodies in a 3-fold dilution starting from 100 ⁇ g/mL in 0.1 M sodium bicarbonate overnight at 4°C. Plates were washed with PBS + 0.05% Tween 20 and blocked with 3% BSA in PBS at room temperature for 1 h.
  • K562-E cells and K562-E/UL49.5 cells were resuspended with fresh IMDM media with 10% FBS at 2x106 cells/ml. Peptides were added into cell suspension at a final concentration of 100 ⁇ M. The cell/peptide mixtures were incubated at 26°C with 5% CO2 for 20-22 hours and were transferred to 37°C for 2 hours with 5% CO2 before use. In the following mAb staining experiment, medium with 100 ⁇ M peptides was used to maintain peptide concentration. [0430] NK Cell Cytotoxicity Assay [0431] NK Cell Cytotoxicity was measured by 51Cr release assay.
  • a NKG2A-positive, CD16/CD32/CD64-negative NK-92 cells were used as effector cells in our study.
  • Transfected or untransfected 293T cells were used as target cells.
  • Target cells were counted, washed, resuspended in R10 at 1 ⁇ 107 cell/ml, and labeled with Na251CrO4 at 250 ⁇ Ci/ml for 2 hours at 37°C. After washing three times using R10, cells were mixed with the testing antibody and effector cells in a final effector to target (E:T) ratio of 20:1 and 6:1 in triplicate wells in a flexible 96 well round bottom plates (PerkinElmer, Catalog# 1450-401).
  • the plates were inserted in flexible 96-well plate cassettes (PerkinElmer, Catalog# 1450-101), sealed and incubated at 37°C for 4 hours. After the incubation, cells were pelleted by centrifugation, and from the top of the well, add 25 ul of supernatant to a rigid 96 well isoplates (PerkinElmer, Catalog#1450-514) containing 150 ul of Ultima Gold LSC Cocktail (Sigma, Catalog# L8286).
  • the plates were inserted in rigid 96-well plate cassettes (PerkinElmer, Catalog# 1450-105), sealed and counted on Perkin Elmer Microbeta Triux 1450 counter.51Cr labeled target cells without effector cells were set as a spontaneous release control, and 51Cr labeled target cells mixed with detergent (2% Triton X 100) were used as a maximum release control.
  • PCR products were gel extracted (Qiagen Gel Extraction kit) to select full length genes as per the manufacturer’s protocol.3H4 scFv variants were displayed in library format on the surface of yeast as previously described (Benatuil et al., 2010; Chao et al., 2006). Briefly, S. cerevisiae EBY100 cells were transformed by electroporation with a 3:1 ratio of 12 ⁇ g scFv library DNA and 4 ⁇ g pCTcon2 plasmid digested with BamHI, SalI, NheI (New England Biolabs). The size of the transformed library, determined by serial dilution on selective plates, was 5x107 individual colonies.
  • Yeast Libraries were grown in SDCAA media (Teknova) supplemented with pen-strep at 30°C and 225 rpm.80% of the sequences recovered from the transformed libraries were confirmed to contain full length, in-frame genes by Sanger sequencing (Genewiz). scFv expression on the surface of yeast was induced by culturing the libraries in SGCAA (Teknova) media at a density of 1x107 cells/mL for 24-36 hours.
  • Double positive cells for APC and FITC were collected and expanded in SDCAA media supplemented with pen-strep before successive rounds of enrichment.
  • FACS data was analyzed with Flowjo_v10.7 software (Becton, Dickinson & Company). All clones selected by FACS were expanded, and their DNA was extracted (Zymo Research) for analysis by Sanger sequencing (Genewiz).
  • scFv encoding plasmids were recovered from yeast cultures by yeast miniprep with the Zymoprep yeast plasmid miniprep II kit (Zymo Research). Isolated DNA was transformed into NEB5 ⁇ strain of E.
  • HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795-799.
  • Example 2 Optimization of HLA-E-VL9 binding antibodies
  • the following antibody properties could be targeted for improvement: 1) Binding affinity for the HLA-E/VL9 peptide with an improved dissociation constant, e.g.
  • the invention contemplates computational optimization methods which could include without limitation the following: Identify mutations to increase binding affinity, alter CDR loop length/sequence for increased affinity and/or target specificity.
  • Antibody libraries could include without limitation single site libraries, CDR loops libraries, saturation libraries, directed libraries, structurally informed and/or computationally informed optimization libraries.
  • Any suitable library screening platform could be used. Platforms include without limitation yeast display which allows for screening of scFv, but has limited ability to select for stability/solubility. Platforms include without limitation mammalian display which allows for screening of IgG, assessing expression levels which could correlate with stability and solubility.
  • screening for optimized antibodies include strategies to efficiently select for improved affinity, specificity, reduced polyreactivity, and improved stability.
  • the HLA-E-VL9 binding antibodies will be subjected to multiple rounds of optimization using high throughput screening.
  • libraries of antibody variants, constructed as scFVs and displayed on the surface of yeast will be screened and sorted by FACS in succession as described in Figure 9: 1) five positive selections rounds for binding to the HLA-E/VL9 complex present at a concentration of 500nM in the first round, and two fold dilutions afterwards (250nM, 125nM, 62.5nM, 31.2nM and 16nM respectively); 2) two counter selection rounds for specificity in the presence of the HLA-E/VL9 complex at a concentration of 10nM and HLA-E/RL-9 competitor peptide present at 100nM; 3) two counter selection rounds to eliminate clones that bind heparin, Hsp70 and Hsp90 to eliminate polyreactive clones.
  • the library is a single site CDR loop saturation library, e.g. designed to contain all the twenty amino acids variants at each position in the CDR loops, mutated one at a time.
  • the libraries are site directed libraries that explores all possible amino acid variants only at the antibody sites involved in binding interactions with HLA-E/VL9, for example as informed from the crystal structure of the complex.
  • the yeast display platform is well suited to analyze 10 antibody variants at a time; therefore all the possible amino acids combinations will be simultaneously tested at groups of four antibody residues to ensure that the number of resulting antibody variants to be screened experimentally is below 10 7 .
  • 3H4 optimization and libraries based on HLA-E-VL9/3H4 complex structure [0482] The 3H4 antibody has four key contacts with the HLA-E/VL9 peptide complex. See Figure 2. These contacts are Y97, S100, S100A and Y100B. [0483] Additional antibody residues/sites were chosen for modification and antibody optimization. Residues that are chosen to be randomized as part of directed libraries are shown in the structure to contact HLA-E. Based on the analysis of the structure we chose to randomize 7 groups of 4 residues. The groups of four residues are picked such they are close in space and contact the same site of the HLA-E/VL9 complex.
  • the four residues are selected such that they interact with the same region(s) of the epitope and thus may have combined binding effects (Figure 10).
  • Four residues are randomized at a time because this is the number of variants that can be tested by yeast display. Some of the residues in the libraries are part of the CDR H3, but other residues beyond the CDR H3 will also be tested.
  • not all the amino acids in a group will need to be changed during the optimization.
  • any one residue from a group of residues or a combination thereof could be changed in an optimized variant.
  • the combination is a combination of residues within a group.
  • the combination is a combination of residues from different groups.
  • scFv different directed libraries that sample all the amino acid variants at four residues are generated. These libraries aim to test all the 20 amino acid combinations at the four residues shown in bold and red in the sequence in Figure 11.
  • 3H4 SD4 sequence shows one embodiment of a scFv fragment where VH is italicized and VL is double underlined, and HCDR3 sequence is underlined. by alignment to the other variants in Figure 11, the VH, VL, and HCDR3 regions can be determined.
  • the linker between the VH and VL sequence could be any suitable linker of varying length and/or sequence.
  • Figure 11 shows non-limiting embodiments of scFv variants comprising contact residues which could be modified in an optimized variant. Based on structural analysis, seven scFv different directed libraries that sample all the amino acid variants at four sites were designed. The four sites are shown in bold and red in the sequence. Sequence changes identified from these libraries that improve the properties of 3H4 mAb will be further combined in additional libraries that will be screened as in Figure 9 to identified sets of mutations that are cumulative towards the optimization 3H4. Each sequence shows one embodiment of a scFv fragment where VH is italicized and VL is double underlined, and HCDR3 sequence is underlined.
  • the linker between the VH and VL sequence could be any suitable linker of varying length and/or sequence.
  • the optimized antibody sequences will be tested for their binding. Binding affinity will be determined. Binding assays include without limitation cell surface staining, SPR and ELISA.
  • Binding assays include without limitation cell surface staining, SPR and ELISA.
  • HLA-E SCT constructs encoding HLA-E-VL9, HLA-E-RL9HIV, or HLA-E-RL9SIV are transfected into 293T cells using GeneJuice transfection reagent (Novagen, Catalog# 70967).
  • HLA-E-VL9 SCT constructs with single amino acid mutations are transfected into 293T cells using the same method.
  • Cells are dissociated with 0.1% EDTA at 48 hours post-transfection and stained with a Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, Catalog# L34976).
  • primary antibodies supernatant from hybridoma cells, supernatant from transfected cells, or purified antibodies
  • Alexa Fluor 555 conjugated goat anti- mouse IgG (H+L) (Thermo Fisher, Catalog# A32727) is used or Alexa Fluor 647 (AF647) conjugated goat anti-mouse IgG (H+L) (Thermo Fisher, Catalog# A32728) as secondary antibodies;
  • AF555 conjugated goat anti-human IgG (H+L) (Thermo Fisher, Catalog# A-21433) is used or AF647 conjugated goat anti-human IgG (H+L) (Thermo Fisher, Catalog# A-21445) as secondary antibodies.
  • HLA-E- VL9, HLA E RL9SIV, HLA E RL9HIV and mock control biotinylated HLA-E-peptide complexes
  • Direct binding ELISAs were conducted in 384-well ELISA plates coated with 2 ⁇ g/ml of C-trap-stabilized HLA-E-VL9, C-trap-stabilized HLA-E-RL9HIV or C-trap- stabilized HLA-E-RL9SIV in 0.1 M sodium bicarbonate overnight at 4°C. Plates were washed with PBS + 0.05% Tween 20 and blocked with 3% BSA in PBS at room temperature for 1 h. MAb samples were incubated for 1 h in 3-fold serial dilutions starting at 100 ⁇ g/ml, followed by washing with PBS-0.05% Tween 20.
  • HRP-conjugated goat anti-human IgG secondary Ab (SouthernBiotech, catalog# 2040-05) was diluted to 1: 10,000 in 1% BSA in PBS-0.05% Tween 20 and incubated at room temperature for 1 h.
  • sandwich ELISA 384- well ELISA plates were coated with HLA-E-VL9 antibodies in a 3-fold dilution starting from 100 ⁇ g/mL in 0.1 M sodium bicarbonate overnight at 4°C. Plates were washed with PBS + 0.05% Tween 20 and blocked with 3% BSA in PBS at room temperature for 1 h.
  • NK Cell Cytotoxicity assays [0493] To test if an optimized antibody has higher killing function, we will perform NK Cell Cytotoxicity Assay by 51 Cr release assay. Human NK-92 cells are used as effector cells in our study. Transfected 293T cells are used as target cells. Target cells are counted, washed, resuspended in R10 at 1 ⁇ 10 7 cell/ml, and labeled with Na 2 51 CrO 4 at 250 ⁇ Ci/ml for 2 hours at 37°C.
  • the plates are inserted in rigid 96-well plate cassettes (PerkinElmer, Catalog# 1450-105), sealed and counted on Perkin Elmer Microbeta Triux 1450 counter.
  • 51 Cr labeled target cells without effector cells are set as a spontaneous release control
  • 51 Cr labeled target cells mixed with detergent 2% Triton X-100
  • AtheNA assay All mAbs isolated from mice and human are tested for ELISA binding to nine autoantigens - Sjogren's syndrome antigen A (SSA), Sjogren's syndrome antigen (SSB), Smith antigen (Sm), ribonucleoprotein (RNP), scleroderma 70 (Scl-70), Jo-1 antigen, double-stranded DNA (dsDNA), centromere B (Cent B), and histone as previously described (Han et al., 2017; Liao et al., 2011).
  • Size-exclusion Chromatography (SEC) Assay Antibodies will be flowed through a column consisting of spherical beads with miniscule pores. Non-aggregated antibodies will be small enough to get trapped in the pores, whereas aggregated antibodies will flow through the column more rapidly. Percentage aggregation can be worked out from the concentrations of the different fractions.
  • CSI-BLI Clone Self-interaction by Bio-layer Interferometry
  • CSI-BLI A more high- throughput method that uses a label-free technology to measure self-interaction. Antibodies will be loaded onto the biosensor tip and white light is shone down the instrument to yield an internal reflection interference pattern.
  • Affinity-capture Self-interaction Nanoparticle Spectroscopy (AC-SINS) Assay This assay tests how likely an antibody is to interact with itself. It uses gold nanoparticles that are coated with anti-Fc antibodies. When a dilute solution of antibodies is added, they rapidly become immobilised on the gold beads. If these antibodies subsequently attract one another, it leads to shorter interatomic distances and longer absorption wavelengths that can be detected by spectroscopy. [0500] See e.g. Jain et al.
  • Such contact and proximal residues could be organized in groups of 4 residues.
  • the groups of four residues are picked such they are close in space and contact the same site of the HLA-E/VL9 complex.
  • the four residues are selected such that they interact with the same region(s) of the epitope and thus may have combined binding effects).
  • Four residues are randomized at a time because this is the number of variants that can be tested by yeast display.
  • Some of the residues in the libraries are part of the CDR H3, but other residues beyond the CDR H3 will also be tested. [0505]
  • not all the amino acids in a group will need to be changed during the optimization.
  • any one residue from a group of residues or a combination thereof could be changed in an optimized variant.
  • the combination is a combination of residues within a group. In some embodiments the combination is a combination of residues from different groups.
  • scFv different directed libraries that sample all the amino acid variants at four residues are generated. These libraries aim to test all the 20 amino acid combinations at the four residues shown in Figure 11. Residues could be selected from VH and/or VL residues. Residues could be within CDRs or outside of the CDRs. In scFv designs, the VH and VL are connected by a linker. The linker between the VH and VL sequence could be any suitable linker of varying length and/or sequence. See e.g. Chao et al.
  • scFv libraries currently being built and/or screened in yeast [0510] CA147 (anti-HLA-E/VL9) single-site saturation (NNK) library of all CDR loops [0511] CA123 (anti-HLA-E/VL9) single-site saturation (NNK) library of all CDR loops [0512] Any other human HLA-E/VL-9 antibody could be optimized.
  • the binding strength between antibodies and their cognate antigen is increased by providing avidity to the interaction with either by the presence of multiple B cell receptor on the surface of B cells or through the formation of dimers, pentamers, and hexamers of secreted immunoglobulin (Czajkowsky et al Proc Natl Acad Sci USA 2009; Kumar et al. Science.2020). Similar principles can be applied to antibody biologics. Through avidity, we sought to increase the binding strength of these antibodies thereby enhancing their ability to mediate killing of infected cells.
  • Each engineered antibody was expressed by transient transfection of Expi293F cells and purified by anti-kappa or anti-lambda constant region affinity resin. SDS-PAGE analysis showed the presence of heavy and light chains. To assess the presence of hexamer formation, we performed negative stain electron microscopy on each protein preparation. Electron microscopy confirmed the presence of some monomeric IgG, but also hexameric IgG ( Figure 25B, C). The hexameric IgG was purified by size exclusion chromatography to enrich for IgG hexamers.
  • Another non-limiting approach to form multimeric antibody is to generate IgG antibodies or fragments thereof arrayed in multiple copies on the surface of nanoparticles, including without limitation protein nanoparticles.
  • the nanoparticle is a ferritin nanoparticle.
  • Antibody nanoparticles were generated as conjugate nanoparticles by adding a sortase A donor peptide (also referred to a sortase tag or linker) to the C-terminus of the heavy chain constant region and by adding a sortase acceptor sequence to the N-terminus of each ferritin nanoparticle subunit.
  • a sortase A donor peptide also referred to a sortase tag or linker
  • a sortase acceptor is added to the N-terminus of the heavy or light chain constant region and a sortase donor peptide sequence to the C- terminus of each ferritin nanoparticle subunit.
  • Any suitable heavy chain constant region can be used.
  • the donor peptide can vary at the third position, but A, E, and S tend to be the most common amino acids used.
  • the acceptor sequence can be 5 or more glycines.
  • the multimer antibody was designed as a full-length IgG.
  • the multimer was designed as an antigen binding fragments (Fabs).
  • the sortase tag is added to the C-terminus end of the Fab heavy chain sequence ( Figure 26).
  • the sortase tag is added to the C- terminus of the Fab light chain sequence.
  • Any suitable constant gene could be used for the design of full length IgG, or the Fab fragment.
  • One non-limiting embodiment of a Fab design is shown in Figure 26A. [0519] The Fabs of each antibody expressed well in Expi293 cells. The sortase A-tagged Fab molecule was conjugated to ferritin nanoparticles overnight at room temperature in the presence of 100 ⁇ M recombinant sortase A. Size exclusion chromatography was used to separate Fab-nanoparticle conjugates. SDS-PAGE analysis of Fab conjugate nanoparticles confirmed the Fab molecule was conjugated to ferritin subunits.
  • HLA E single chain trimer constructs encoding HLA E VL9 or HLA E Mtb44 were transiently transfected into 293T cells using GeneJuice transfection reagent (EMD Millipore, Catalog# 70967). Two days post transfection, cells were diassociated with 1mM EDTA and were washed and resuspended in 1x PBS pH 7.4.
  • the multimeric antibodies or fragments thereof will be tested in any suitable assay to characterize their properties, including without limitation any of the assays described herein.
  • Example 4 Optimization of HLA-E-VL9 binding antibodies – including non-limiting embodiments of affinity matured 3H4 antibodies.
  • 3H4 scFv library was transformed into yeast and screened for three rounds by fluorescence-activated cell sorting (FACS) for binding to fluorescently labeled HLA-E-VL9 tetramer. Twelve 3H4 variants (v1-v12) were selected for experimental characterization as recombinant human IgGs from the highly represented clones remaining in the library upon the final selection round. These novel Abs (3H4G_v1 to 3H4G_v12) were mutated at positions 97-100 of the CDR H3 loop.
  • the optimized antibodies Compared to the original 3H4 mAb, the optimized antibodies predominantly contained small amino acids at positions 97 and 98, a polar amino acid at position 99, and a large aromatic at position 100 that is closest to the HLA-E-VL9.
  • additional libraries were generated by sampling all the possible combinations of amino acids at positions 101, 102 and 103 in the CDR H3 loops of the mAbs that had the highest affinities for HLA E VL 9 (3H4G_v3, 3H4G_v5 and 3H4G_v6). These libraries were screened on the surface of yeast by FACS as above for binding to HLA-E-VL9.
  • Second generation antibodies were isolated that originated from each of 3H4G_v3, 3H4G_v5 and 3H4G_v6 and that contained additional mutations at positions 101-103 in the CDR H3 loop. These second-generation antibodies, 3H4G_v31, 3H4G_v51, 3H4G_v61 and 3H4G_v62 had 2-4 times higher affinity for HLA-E- VLp compared to the first generation optimized mAbs.
  • Example 5 Anti-HLA-E/VL9 affinity-matured antibodies are active in vivo.
  • mice were injected with K562 myelogenous leukemia cells pre-transfected with HLA- E/VL-9 complexes at day 0.
  • mice that developed tumors were subjected to treatment at days 10, 14, 16 and 18 with the further affinity matured 3H4 antibody 3H4G_v31 in combination with NK-92 natural killer cells and IL-12 to support NK cell function.
  • CH65 an anti-influenza antibody, was used as control.
  • Tumor size was assessed daily and animals were euthanized once tumor sizes exceeded the approved threshold.
  • Retarded HLA-E/VL-9+ tumor growth was seen in the presence of affinity-matured 3H4 antibody and NKG2A+ NK cells ( Figure 32). Each curve represents a mouse and the grey arrows indicate treatment. Tumor size was measured every day and the volume (mm 3 ) was calculated as length x width x height. CH65 is a control mAb (anti-influenza). Increased NSG mouse survival was seen in the presence of affinity-matured 3H4 antibody and NKG2A+ NK cells ( Figure 33).
  • SPR assay screening was performed. For manual screening of HLA-E-VL9 proteins, a Q-injection of 90 ⁇ l of HLA-Ebt-VL9 MNQ protein at concentration of 50 ⁇ g/mL was performed over the immobilized antibodies at a flow rate of 30 ⁇ l/min in HBS-EP+ 1x running buffer. After dissociating, the antibodies were regenerated using 10 ⁇ l of Glycine pH2.0 at a flow rate of 50 ⁇ L/min. ⁇ Cat_Ab82_AAA surface was used for reference subtraction. [0533] SPR assay kinetics were measured.
  • HLA-E-VL9 For injection of HLA-E-VL9 proteins, a K- injection of 90 ⁇ l of HLA-E-VL9 MNQ protein at concentration of 0.5 ⁇ g/ml – 50 ⁇ g/ml was performed over the immobilized antibodies at a flow rate of 30 ⁇ l/min in HBS-EP+ 1x running buffer. No regeneration was necessary. Cat_Ab82_AAA surface was used for reference subtraction. HLA-E-VL9 kinetics on 3H4 IgG variants are shown in Figure 38. Rate constants (ka, kd) and dissociation constant KD were determined by curve fitting analysis of SPR data with 1:1 binding model.
  • Rate constants (ka, kd) and dissociation constant KD were determined by curve fitting analysis of SPR data with 1:1 binding model.
  • Rate constants (ka, kd) and dissociation constant KD were determined by curve fitting analysis of SPR data with 1:1 binding model.
  • Example 7 Binding specificity of affinity matured 3H4 mAbs [0536] Affinity matured 3H4 mAbs were highly specific for HLA-E/VL9 ( Figure 42). HEK 293t cells were transfected with HLA-E and peptides from HLA-E, RL9HIV R1V, SARS- CoV-2001, Mtb and Mamu-E. These peptides can form a complex with HLA-E. The cells were then analyzed by FACS for binding to the optimized 3H4G_v31 and 3H4G_v61.
  • the affinity matured 3H4 mAbs were highly specific for HLA-E/VL9.
  • the wildtype 3H4 bound to four out of five HLA-E/VL9 complexes tested while 3H4G_v61 was only bound to HLA-E/VL-9 as desired.

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Abstract

La présente invention concerne des anticorps monoclonaux recombinants à maturation d'affinité (mAb) et des fragments qui se lient spécifiquement à un complexe HLA-E-peptide, y compris des complexes HLA-E-VL9, et régulent la fonction de cellule effectrice de cytotoxicité de cellules NK et/ou de lymphocytes T CD8 + positives pour l'expression en surface cellulaire de NKG2A ("NKG2A+"). Des anticorps monoclonaux ont été dérivés de manière recombinante de MAb spécifiques de HLA-E-VL9 fonctionnels isolés issus de souris transgéniques HLA-B immunisées contre un peptide HLA-E-VL9 et du répertoire des lymphocytes B humains naïfs. De tels anticorps sont peuvent réguler la cytotoxicité des cellules effectrices et peuvent de préférence reconnaître des complexes peptidiques HLA-E-VL9 exprimés sur la surface de cellules tumorales. Les anticorps monoclonaux ont été soumis à un ou à plusieurs cycles de maturation d'affinité. L'invention concerne des méthodes d'utilisation de mAb HLA-E-VL9 à maturation d'affinité pour moduler la fonction de cellules NK et/ou de lymphocytes CD8+ dans le cadre de stratégies immunothérapeutiques.
EP22859437.0A 2021-08-20 2022-08-19 Anticorps ciblant des complexes peptidiques hla-e-hôte et leurs utilisations Pending EP4388012A1 (fr)

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