WO2026038164A1 - Humanization and affinity maturation of the cd4-mimicking llama nanobody j3 - Google Patents

Humanization and affinity maturation of the cd4-mimicking llama nanobody j3

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Publication number
WO2026038164A1
WO2026038164A1 PCT/IB2025/058241 IB2025058241W WO2026038164A1 WO 2026038164 A1 WO2026038164 A1 WO 2026038164A1 IB 2025058241 W IB2025058241 W IB 2025058241W WO 2026038164 A1 WO2026038164 A1 WO 2026038164A1
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WIPO (PCT)
Prior art keywords
seq
optionally
antigen binding
binding molecule
amino acid
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PCT/IB2025/058241
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French (fr)
Inventor
Wei Li
Zhiwei FENG
Tianjian LIANG
Seungmin SHIN
Margaret Grace HINES
John W. Mellors
Zeyu SUN
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University of Pittsburgh
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University of Pittsburgh
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Publication of WO2026038164A1 publication Critical patent/WO2026038164A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site

Definitions

  • ART antiretroviral therapies
  • Antibody therapeutics are one such therapeutic modality that may be capable of filling this gap in treatment.
  • An antibody’s high affinity for its particular antigen allows these treatments to effectively target specific cells and reduce potential off-target effects of the therapy.
  • a humanized antigen binding molecule including a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20.
  • HIV human immune deficiency virus
  • a method of binding or neutralizing a human immune deficiency virus (HIV) particle including contacting the HIV particle with a humanized J3 derivative antigen binding molecule.
  • a method of treating a patient infected with HIV including administering to the patient a humanized J3 derivative antigen binding molecule to the patient in an amount effective to treat the infection.
  • a composition including a humanized J3 derivative antigen binding molecule in a pharmaceutically-acceptable excipient, vehicle, or carrier is also provided herein.
  • a CAR-T cell expressing an antigen binding molecule having the amino acid sequence of SEQ ID NO: 14.
  • a CAR-T cell expressing an antigen binding molecule having the amino acid sequence of SEQ ID NO: 3.
  • a humanized antigen binding molecule comprising a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 3 66H6137.
  • DOCX Attorney Docket No.06527-2502271 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0030] 12.
  • a method of binding or neutralizing a human immune deficiency virus (HIV) particle comprising contacting the HIV particle with a humanized J3 derivative antigen binding molecule.
  • the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14.
  • the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14.
  • 15 The method of any of clauses 12-14, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14.
  • the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [0038] 20. The method of any of clauses 12-19, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. [0039] 21. The method of any of clauses 12-20, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. [0040] 22.
  • the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID 4 66H6137.
  • DOCX Attorney Docket No.06527-2502271 NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0041] 23.
  • the HIV particle is an HIV1 particle.
  • 24 A method of treating a patient infected with HIV comprising administering to the patient a humanized J3 derivative antigen binding molecule to the patient in an amount effective to treat the infection.
  • 25 The method of clause 24, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14.
  • 26 The method of clause 24 or clause 25, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. [0045] 27.
  • composition of any of clauses 36-44, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9.
  • the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID 6 66H6137.
  • DOCX Attorney Docket No.06527-2502271 NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20.
  • FIG. 1 shows the workflow of the IsAb2.0; users are required to input the sequences of antibody and antigen into IsAb2.0 protocol; AlphaFold-Multimer 2.3/3.0 generates 3D structure of antibody–antigen complex based on their sequences; if the prediction quality (pLDDT) of complex is below 70, the structure undergoes refinement; SnugDock is then used to refine the antibody–antigen complex, followed by alanine scanning and FlexddG for hotspot identification and antibody optimization.
  • FIG. 1 shows the workflow of the IsAb2.0; users are required to input the sequences of antibody and antigen into IsAb2.0 protocol; AlphaFold-Multimer 2.3/3.0 generates 3D structure of antibody–antigen complex based on their sequences; if the prediction quality (pLDDT) of complex is below 70, the structure undergoes refinement; SnugDock is then used to refine the antibody–antigen complex, followed by alanine scanning and FlexddG for hotspot identification and antibody optimization.
  • FIG. 2 shows comparison between HuJ3-gp120 and J3-gp120 3D structures.
  • the general binding poses of HuJ3 and J3 with gp120 are highly similar, especially their CDR loops.
  • FIG. 3 shows comparison of hydrogen bonds between HuJ3 and J3 with gp120. Yellow dash line is hydrogen bond.
  • (A) and (B) are the hydrogen bonds on HuJ3-gp120.
  • C) and (D) are the hydrogen bonds on J3-gp120. Most of the residue pairs forming hydrogen bonds are the same between HuJ3-gp120 and J3-gp120 complexes.
  • FIGS. 4A-4C show comparison of hydrogen bonds between HuJ3 and designed HuJ3 with gp120. The yellow dashed line is the hydrogen bond.
  • E44R mutation R44 enhances electrostatic interactions favorably.
  • V54E mutation E54 forms two hydrogen bonds with R432 compared with V54.
  • S98W mutation W98 increases the electrostatic property of the HuJ3-gp120 complex.
  • FIG.5 shows experimental validation of designed HuJ3 variants.
  • A Binding affinity of HuJ3 variants predicted by IsAb1.0 for HIV-1 Bal gp140.
  • B Binding affinity of HuJ3 variants predicted by IsAb2.0 for HIV-1 Bal gp140.
  • FIG.6 shows alignment of the HuJ3-gp120 complexes generated by IsAb1.0 and IsAb2.0.
  • FIG. 7 shows sequences of various nanobodies, including HuJ3 (SEQ ID NO: 1) and NanoJ3 (SEQ ID NO: 2), according to non-limiting embodiments disclosed herein.
  • FIG.8 shows sequences of various nanobodies, including full-length HuJ3 (SEQ ID NO: 14) and 4E2 (SEQ ID NO: 3), according to non-limiting embodiments disclosed herein. [0080] FIG.
  • FIG. 10 shows sequences of various nanobodies, including full-length NanoJ3 (SEQ ID NO: 15), 4E2 (SEQ ID NO: 3), full-length HuJ3 (SEQ ID NO: 14), and G12 (SEQ ID NO: 9), according to non-limiting embodiments disclosed herein.
  • FIG.11 shows the sequence alignment of 5 designed mutations by IsAb2.0, including E44R (SEQ ID NO: 16), V54E (SEQ ID NO: 10), S98W (SEQ ID NO: 11), T99R (SEQ ID NO: 12), and S102R (SEQ ID NO: 13).
  • the amino acids marked as black are the amino acids being mutated compared with HuJ3 (SEQ ID NO: 1).
  • polymer molecular weight is expressed as number-average molecular weight (Mn).
  • Mn number-average molecular weight
  • contacting refers to placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell. In other examples, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample.
  • An “isolated” or “purified” biological component refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in the cell of the organism in which the 9 66H6137.DOCX Attorney Docket No.06527-2502271 component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified”, thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods.
  • an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel.
  • a preparation may be purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.
  • a nucleic acid molecule refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide.
  • the term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA.
  • a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • a first nucleic acid is said to be operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid.
  • operably linked DNA sequences are contiguous (e.g., in cis) and, where the sequences act to join two protein coding regions, in the same reading frame (e.g., open reading frame or ORF), for example to produce a fusion protein.
  • Operably linked nucleic acids include a first nucleic acid contiguous with the 5′ or 3′ end of a second nucleic acid.
  • a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid.
  • a “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species).
  • nucleic acid 10 66H6137.DOCX Attorney Docket No.06527-2502271 sequence can be optimized for expression in yeast cells. Codon optimization does not alter the amino acid sequence of the encoded protein.
  • a conservative substitution is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide.
  • an antigen binding molecule or antibody polypeptide sequence may include one or more conservative substitutions (for example 1-10, 2-5, or 10-20, or no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) yet retains the affinity or avidity of a given antigen binding molecule such as those described herein for binding to, among other possibilities, human immunodeficiency virus (HIV).
  • a polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Methods are provided herein to ascertain proper expression of any sequence.
  • a polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used.
  • the terms “polypeptide”, “peptide”, or “protein” as used herein are intended to encompass any amino acid sequence and include proteins and modified sequences such as glycoproteins.
  • the term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are synthetically produced such as by recombinant or chemical synthesis methods.
  • the term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.
  • Conservative amino acid substitutions are those substitutions that, when made, least or minimally interfere with the properties of the original protein, that is, in the context of the end-use, the structure and function of the protein is conserved and not significantly changed by such substitutions, and may be identified by use of matrices, such as the BLOSUM series of matrices, and other matrices.
  • Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • epitope refers to a physical structure or moiety of a molecule that interacts with an antibody or antibody binding reagent.
  • the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications, can define an epitope.
  • HIV protein and protein fragments including isoforms and post-transcriptionally-modified polypeptides for use in the immunodetection methods, devices, and kits may be produced in mammalian cells, such as HEK293 cells, to produce a protein with mammalian post-translational modifications.
  • Portions of a natural protein can contain an epitope present in the complete natural protein and typically react to antibodies raised to the natural protein.
  • a recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids.
  • sequence identity refers to the similarity between nucleic acid or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity may be measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, isoforms, or variants of a polypeptide often possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well- known in the art.
  • the percent sequence identity value may be rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants may typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. [0099] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences may be entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters 13 66H6137.DOCX Attorney Docket No.06527-2502271 may be used. Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, for example and without limitation, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithm of Needleman & Wunsch, by the search for similarity method of Pearson & Lipman, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by manual alignment and visual inspection.
  • GAP Garnier et al.
  • BESTFIT Pearson & Lipman
  • FASTA Pearson & Lipman
  • TFASTA Pearson & Lipman
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle.
  • a reference sequence may be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package (see, e.g., Chao J, et al. Biomolecules.2022 Apr 6;12(4):546).
  • Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program may be used for nucleotide sequences.
  • the BLASTP program may be used for amino acid sequences.
  • reference to “at least 70% identity” may refer to “at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.
  • reference to “at least 90% identity” may refer to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.
  • Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide strands that are typically in antiparallel orientation.
  • Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • uracil rather than thymine is the base that is complementary to adenosine.
  • Two sequences 14 66H6137.DOCX Attorney Docket No.06527-2502271 comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1X SSC (saline sodium citrate) to 10X SSC, where 1X SSC is 0.15M NaCl and 0.015M sodium citrate in water.
  • saline e.g., normal saline, or 0.9% w/v saline
  • phosphate-buffered saline phosphate-buffered saline
  • stringency conditions such as, for example and without limitation, 0.1X SSC (saline sodium citrate) to 10X SSC, where 1X SSC is 0.15M NaCl and 0.015M sodium cit
  • a vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • An insertional vector is capable of inserting itself into a host nucleic acid.
  • a vector can also include one or more selectable marker genes and other genetic elements.
  • An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. [00104] By “expression” or “gene expression,” it is meant the overall flow of information from a gene.
  • a “gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as an ncRNA that has a function.
  • a “gene” is a functional genetic unit for producing its gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and generally comprising: a transcriptional control sequence, such as a promoter and other cis- acting elements, such as transcriptional response elements (TREs) and/or enhancers; an expressed sequence that typically encodes a protein (referred to as an open- reading frame or ORF) or functional/structural RNA; and a polyadenylation sequence).
  • a transcriptional control sequence such as a promoter and other cis- acting elements, such as transcriptional response elements (TREs) and/or enhancers
  • an expressed sequence that typically encodes a protein referred to as an open- reading frame or ORF
  • functional/structural RNA referred to as an open- reading frame or functional/structural RNA
  • a gene produces a gene product (typically a protein, optionally post-translationally modified, or a functional/structural RNA) when transcribed
  • genes under transcriptional control of or alternately “subject to control by” a designated sequence such as a promotor, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene.
  • a gene that is “under transcriptional control” of an inducible promotor or transcription control element is a gene that is transcribed at detectably different levels 15 66H6137.DOCX Attorney Docket No.06527-2502271 in the presence of a transcription factor, e.g., in specific cell types or conditions.
  • a “gene for expression of” a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment, that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression.
  • suitable conditions means that the gene typically need only be introduced into a host cell.
  • suitable conditions means when factors that regulate transcription, such as DNA-binding proteins, are present or absent, for example, an amount of the respective inducer is available to the expression system (e.g., cell), or factors causing suppression of a gene are unavailable or displaced - effective to cause expression of the gene.
  • Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100–1000 base pairs long. Additional sequences and non-coding elements can affect transcription rates. If the cell has a nucleus (eukaryotes), the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation refers to the addition of a poly(A) tail to a messenger RNA.
  • the poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases.
  • RNA messenger RNA
  • Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide.
  • the 5’ cap (cap-0) found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage.
  • pre-mRNA is 16 66H6137.DOCX Attorney Docket No.06527-2502271 edited. Specifically, during this process introns are removed, and exons are joined together.
  • RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
  • Gene expression involves various steps, including transcription, post- transcriptional RNA modification, translation, and post-translational modification of a protein. Expression of a gene may also include reduction of the total amount of the protein product, such as by cleavage, sequestration, binding, or other means of decreasing the function or amount of a protein product.
  • Nucleic acids and vectors encoding the described fusion proteins may be provided. In some non-limiting examples, disclosed is a recombinant vector, such as a yeast plasmid, that expresses the disclosed fusion proteins.
  • nucleic acids which differ in sequence, but which encode the same protein sequence due to codon degeneracy.
  • the polynucleotide is codon-optimized for expression in mammalian cells.
  • Exemplary nucleic acids may be prepared by cloning techniques, e.g., as are broadly-known and implemented either commercially, or in the art. Multiple textbooks and reference manuals describe and provide examples of useful and appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through such techniques are known. Commercial and public product information from manufacturers of biological reagents and experimental equipment also provide useful information.
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • a wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill. 17 66H6137.DOCX Attorney Docket No.06527-2502271
  • antigen binding molecules e.g., antibody compounds comprising an antibody domain targeting a protein, for example an HIV protein, for example an HIV gp120 protein (including, without limitation, recombinant proteins) and epitopes, and methods of use of those compounds.
  • antigen binding molecule refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan A, Marcatili P, Tramontano A. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 Aug;278(16):2858-66, and US Patent No. 11,578,428 B2, and U.S. Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies).
  • the antigen binding molecule operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein.
  • Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen.
  • the term “VH” refers to a heavy chain variable region of an antibody.
  • VL refers to a light chain variable region of an antibody.
  • Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be “humanized” to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal.
  • An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • An antigen binding molecule or complexes thereof may be, for example and without limitation, a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab′, Fv fragments, single chain Fv (scFv) fragments, dsFv, Fab1 fragments, F(ab′)2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are co
  • Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.
  • scFv molecules may be manufactured using any suitable technology.
  • recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S.
  • the antigen binding molecules described herein comprise, at their core paratopes formed from VL and VH polypeptides, that are defined by three CDR’s (typically loops), CDR1, CDR2, and CDR3, which for VH peptides may be termed HCDR1, HCDR2, and HCDR3, respectively, and which for VL peptides may be termed LCDR1, LCDR2, and LCDR3, respectively, each of which are flanked by, and separated by framework (e.g., joining or scaffold) amino acid sequences that space apart and support the CDRs, and which may differ from antibody-to-antibody, and which may be “humanized” to minimize antigenicity when administered to a human 19 66H6137.
  • DOCX Attorney Docket No.06527-2502271 patient.
  • HCDR3 and LCDR3 are typically the most variable of the CDRs, contributing significantly to antibody specificity.
  • Various methods may be used to identify the precise limits of each CDR, but the sequences provided herein can be evaluated by any suitable method to determine the CDRs.
  • Exemplary sequences of antibody heavy and light chains are provided in the attached sequence listing, which is incorporated herein by reference in its entirety.
  • Antibody constant and variable regions, including CDR sequences and framework sequences can be readily ascertained from the sequences provided in in the attached sequence listing.
  • Reference to a CDR herein may refer to a Kabat CDR numbering scheme or any other applicable CDR numbering scheme, including but not limited to Chothia, Martin (enhanced Chothia), Gelfand, IMGT, Honneger, or any other numbering scheme (see, e.g., Dondelinger M, et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol. 2018 Oct 16;9:2278).
  • antibody sequence annotation, including definition of CDR sequences may be conducted using tools described and provided in abYsis (abysis.com), or Abnum (www.bioinf.org.uk/abs/abnum/).
  • CDR identification is known in the art (see, e.g., Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4; Adolf- Bryfogle J, Xu Q, North B, Lehmann A, Dunbrack RL Jr. PyIgClassify: a database of antibody CDR structural classifications. Nucleic Acids Res. 2015 Jan;43(Database issue):D432-8), and assorted online tools and applications as are broadly-available.
  • an amino acid sequence of a VH or VL may be provided or determined, comprising CDRs, and one of ordinary skill can determine the precise metes and bounds of CDRs within that antibody sequence without undue experimentation.
  • Framework sequences may be optimized (see, e.g., Gopal R, Fitzpatrick E, Pentakota N, Jayaraman A, Tharakaraman K, Capila I. Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti- SARS-CoV-2 Antibody.
  • Antibodies may be produced by any effective method, such as by hybridoma or it may be recombinantly or synthetically produced.
  • antibodies may refer to both natural antibodies as well as protein antibody analogs, antibody fragments, and derivatives, any of which comprising VL and/or VH sequences and/or CDRs (e.g., all three CDRs of any VH or VL region, defining a paratope as described herein) according to any example, aspect, or embodiment described herein.
  • the antibody or antibodies may be synthetic, in that they do not comprise a naturally-occurring sequence, such as certain antigen binding molecules, including engineered versions and derivatives thereof, such as scFv versions thereof, humanized versions thereof, BiTEs, and/or sequence derivatives thereof, including without limitation, proteins comprising the CDRs (e.g., one or more, or all three CDRs) of the antibodies provided herein.
  • Nanobodies which may be referred to a VHH antibodies or single-domain antibodies, may be constructed using CDR sequences, such as CDRs of the antibodies described herein.
  • Nanobodies may be created by grafting of the complementarity determining regions (CDRs) from already existing, non-camelid antibodies to VHH frameworks, followed by affinity maturation using synthetic phage libraries (see, e.g., Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two- Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci.2018 Nov 2;19(11):3444).
  • CDRs complementarity determining regions
  • a VH may be capable of defining an antigen-binding site (e.g., paratope) with sufficient strength to be pharmacologically-useful, and can be referred to as a nanobody (see, e.g., Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol.
  • An antibody-drug conjugate may be provided.
  • An antigen binding molecule according to any aspect, embodiment, or example provided described herein may be linked to a payload (e.g., a cargo or warhead) that causes a desired physiological effect, such as killing a cell expressing a binding partner to the antigen binding molecule.
  • the antigen binding molecule e.g.
  • ADCs comprise an antigen binding moiety (a linked antigen-binding molecule), a linker that may be cleavable or non-cleavable, and the payload moiety.
  • a “moiety” is a chemical group or entity, often functional, that forms part of a larger molecule.
  • Linkers may be used to join a payload moiety to the antigen binding molecule, and choice of linkers can depend on how the APC is handled by the cell, and how the payload becomes effective on processing by a cell, or by release due to chemical lability of the linker.
  • Non-cleavable linkers include, without limitation, 22 66H6137.DOCX Attorney Docket No.06527-2502271 alkyl moieties, and thioether moieties (e.g., Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC)).
  • SMCC Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate
  • Cleavable or labile linkers may include, for example and without limitation, acid-labile linkers (hydrolysable in lysosomes or endosomes), Lysosomal protease–sensitive linkers (e.g., peptide-based linkers), ⁇ -glucuronide linkers, and glutathione-sensitive disulfide linkers, with examples including, without limitation: ester-, hydrazone-, Valine-citrulline (v-c)-, Valine-alanine (v-a)-, and phenylalanine-lysine (p-l)-containing linkers.
  • acid-labile linkers hydrolysable in lysosomes or endosomes
  • Lysosomal protease–sensitive linkers e.g., peptide-based linkers
  • ⁇ -glucuronide linkers e.g., glutathione-sensitive disulfide linkers
  • payloads include, without limitation: microtubule-disrupting agents, such as auristatin, maytansinoids, eribulin (e.g., eribulin mesylate), tubulysins, cryptophycins, and EG5 inhibitors; DNA-damaging agents, such as, without limitation calicheamicin, duocarmycins, doxorubicin, enediyne, topoisomerase I inhibitors, and Pyrrolo[2,1- c][1,4] benzodiazepines; RNA-targeting payloads, such as thailanstatins and amatoxins; immune payloads, such as Toll-like receptor agonists, STING agonists, glucocorticoid receptor modulators; and other payloads, such as Bcl-xL inhibitors, NAMPT inhibitors, and proteasome inhibitors such as carmaphycins.
  • microtubule-disrupting agents such as
  • an ADC comprising an antigen binding molecule as described herein linked to a cytotoxic payload via a chemical linker.
  • an antigen binding molecule as described herein linked to a cytotoxic payload via a chemical linker.
  • any antigen binding molecule comprising CDR sequences as described herein 23 66H6137.DOCX Attorney Docket No.06527-2502271 for binding an HIV protein is contemplated.
  • Such antigen binding molecules may be evaluated and used in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Müller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes. Front Mol Biosci. 2020 May 27;7:98), single-molecule array (e.g., SIMOA), surface plasmon resonance (SPR), oblique- incidence reflectivity difference (OI-RD) binding affinity, or cell binding assay, are contemplated.
  • BLItz bilayer interferometry
  • Antigen binding molecules with high binding affinities may bind to their corresponding antigen with a KD of 1 ⁇ M or less, 500 nM or less, 100nM or less, 75nM or less, 50nM or less, or 25 nM or less.
  • Such antigen binding molecules may find use in therapies for conditions related to (e.g., responsive to) MET agonists, and for diagnostic purposes.
  • target-specific or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S. Food and Drug Agency (FDA), in aspects, in the context of administering a reagent as described herein to a patient.
  • FDA U.S. Food and Drug Agency
  • the “treatment” or “treating” of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure (e.g., an antigen binding molecule or antibody as described herein) with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for a condition associated with a viral protein, including an HIV protein, and also includes monitoring the patient by any useful method, including by use of an antigen binding molecule described herein.
  • the condition is HIV and/or AIDS.
  • a “therapeutically effective amount” refers to an amount of a drug product or active agent effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • An “amount effective” for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point.
  • the “amount effective” is preferably safe 24 66H6137.DOCX Attorney Docket No.06527-2502271 - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration.
  • a therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.
  • Examples of an effective amount of an active agent compounded in a delivery vehicle includes from 100 CAR-T cells per kilogram of body weight to 1X10 8 CAR-T cells per kilogram of body weight, including any increment therebetween, 1X10 4 CAR-T cells per kilogram of body weight to 1X10 7 CAR-T cells per kilogram of body weight, such as ranging from 10 3 , from 10 4 , or from 10 5 CAR-T cells to 10 6 , to 10 7 , to 10 8 , to 10 9 , to 10 10 , to 10 11 , or to 10 12 CAR-T cells per dose, including any increment therebetween, such as approximately 10 3 , 10 4 , 10 5 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 CAR-T cells per dose.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate compositions, such as parenteral or inhaled compositions, in dosage unit form for ease of administration and uniformity of dosage.
  • a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc.,
  • an “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose, for example, an amount of an active agent (antigen 25 66H6137.DOCX Attorney Docket No.06527-2502271 binding molecule or antibody) described herein effective to treat a condition described herein.
  • an effective amount of an active agent compounded in a delivery vehicle includes from 1 ⁇ g/ml (micrograms per milliliter) to 100 mg/ml of solution, including any increment therebetween, such as from 1 ⁇ g/ml to 1 mg/ml (milligram/milliliter), all values and subranges therebetween inclusive.
  • Drug products, or pharmaceutical compositions comprising an active agent may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • a “pharmaceutically acceptable excipient”, “carrier”, or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohol’s such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the active agent.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art.
  • Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms.
  • Pharmaceutical formulations adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or 26 66H6137.DOCX Attorney Docket No.06527-2502271 the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts.
  • Pharmaceutical formulations adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion bags, or the like, as are broadly-known to those of ordinary skill.
  • Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution.
  • Pharmaceutical formulations adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery.
  • Pharmaceutical formulations adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization.
  • An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • the therapeutic agents described herein can be administered by any effective route.
  • Examples of delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, 27 66H6137.
  • DOCX Attorney Docket No.06527-2502271 intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances.
  • Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a composition comprising the therapeutic agent useful for treatment of graft rejection as described herein.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • antigen binding molecules such as nanobodies, for binding antigens of interest while minimizing viral escape.
  • the binding molecules described herein can, in non-limiting embodiments, mimic CD4, for example human CD4, thus allowing for effective neutralization of viruses, such as human immunodeficiency virus (HIV), that utilize CD4 as an entry route to a cell.
  • HIV human immunodeficiency virus
  • an antigen binding molecule as described herein may be a J3 derivative antigen binding molecule.
  • a J3 antigen binding molecule may be, in non-limiting embodiments, a nanobody obtained from a camelid (e.g., Camelus dromedarius).
  • the nanobody is obtained from a llama (e.g., Lama glama).
  • the J3 derivative antigen binding molecule may be a humanized binding molecule (e.g., a humanized nanobody).
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 28 66H6137.DOCX Attorney Docket No.06527-2502271 95%, 99%, and/or 100% sequence identity to one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20, all values and subranges therebetween inclusive.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 2, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 3, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 4, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 5, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 6, all values and subranges 29 66H6137.DOCX Attorney Docket No.06527-2502271 therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 7, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 8, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 9, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 10, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 11, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 12, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 13, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 14, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 15, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 16, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 17, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 18, all values and subranges therebetween inclusive.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 19, all values and subranges therebetween inclusive.
  • the sequence may 31 66H6137.DOCX Attorney Docket No.06527-2502271 be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 20, all values and subranges therebetween inclusive.
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • a humanized antigen binding molecule that includes a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), or a sequence having at least 70%, 75%.
  • Antigen binding molecules including humanized antigen binding molecules, as described herein may be used for any purpose, for example, for binding assays (e.g., detection and/or diagnoses) such as immunoprecipitation assays and/or blotting assays (e.g,, Western Blot) as are known in the art.
  • an antigen binding molecule as described herein may be used in prophylactically and/or in treatments, for example to bind and/or neutralize a microorganism, such as a virus (e.g., a virus particle).
  • an antigen binding molecule as described herein may be used to neutralize HIV.
  • Such a method may include contacting an HIV particle with a nanobody as described herein.
  • the HIV particle is an HIV-1 particle.
  • the HIV particle is an HIV-2 particle.
  • the virus is any virus that may enter and/or infect cells via a CD4-mediated pathway.
  • an antigen binding molecule may be formulated for delivery by any suitable route, such as intramuscular, subcutaneous, intraperitoneal, transdermal, and/or the like, with any suitable carriers, vehicles, adjuvants, and/or excipients, and delivered in any suitable concentration/amount for as many doses and/or for as long as is required to provide prophylaxis and/or treatment (e.g., a therapeutically-effective amount).
  • antigen binding molecule may be administered 32 66H6137.DOCX Attorney Docket No.06527-2502271 to a patient in an amount of about 50 to about 100 mg/kg, all values and subranges therebetween inclusive.
  • the method includes administering to a patient an antigen binding molecule, such as a humanized antigen binding molecule, as described herein having an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive.
  • an antigen binding molecule such as a humanized antigen binding molecule, as described herein having an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and
  • the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.
  • the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14.
  • the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3.
  • the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9.
  • the antigen binding molecule useful in a method described herein includes a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), or a sequence having at least 70%, 75%.
  • the antigen binding molecule may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive.
  • the composition may be formulated for parental administration to a patient.
  • CAR chimeric antigen receptor
  • a CAR may include a targeting moiety (e.g., an antigen binding molecule) for binding a surface antigen of another cell, and a signaling moiety for generating a signal when the targeting moiety binds a surface antigen on another cell.
  • a CAR-T cell as described herein may include one or more CARs directed to one or more different surface antigens (target antigens) of a target, for example and without limitation, a B cell, that only activates the CAR-T-cell on which the binding molecules are expressed if the CAR(s) are bound to their target antigen.
  • the CARs may be expressed on the surface of an engineered T cell, e.g., a CAR-T cell, therefore providing a CAR-T receptor that recognizes one or more surface antigens on a B-cell and is only activated when both antigens are present on a target (ABC) cell.
  • target antigens target antigens
  • CAR-T cells to kill B cells or a subset of B cells and/or reducing the population of B cells or a subset of B cells, such as memory B cells, activated B cells, or ABC cells, may be useful in treating a variety of conditions.
  • Relevant conditions include, but are not limited to, HIV, autoimmune diseases, and/or cancers (including, without limitation, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and/or multiple myeloma).
  • CAR-T receptors constructed as earlier described may be designed to target surface antigens from B cells or a subset of B cells.
  • Transfection may be achieved by, for example and without limitation, physical means such as by electroporation, sonoporation, or other methods as are known in the art. Transfection may be achieved by, for example, by chemical means such as by using cationic lipids, cationic polymers, calcium phosphate, DAED-dextran, peptides, nanoparticles, or other methods as are known in the art. Transfection may be of a nucleic acid or nucleic acids comprising a gene or genes for expressing antigen binding molecules, for example as a plasmid or plasmids or with a gene editing system such as a CRISPR Cas9 system.
  • Transduction may be achieved by, for example, lentiviral transduction, wherein a lentivirus inserts a nucleic acid or nucleic acids comprising a gene or genes for expressing CAR-T receptors into the genome of the host cell, such as a T cell. 35 66H6137.DOCX Attorney Docket No.06527-2502271
  • Other recombinant viral-mediated transfection vectors such as AAV, HSV, or Ad vectors, as are broadly-known and available, may be employed for delivery of a nucleic acid comprising a gene for expression in a cell according to the methods described herein.
  • a composition including CAR-T cells may include a pharmaceutically-acceptable excipient or carrier, such as water, PBS, or serum-free cell culture media.
  • a composition may include one or more nucleic acids comprising a gene or genes for expressing one or more antigen binding molecules.
  • a composition may include a plasmid or plasmids comprising a gene or genes for expressing one or more antigen binding molecules.
  • CAR-T cells may be administered in one or more doses and may be administered in an amount effective to reduce the population of B cells or subsets of B cells in a patient. Such amounts may range from, without limitation and inclusive of any increment therebetween, 10 2 to 10 12 CAR-T cells.
  • CAR-T cells may be delivered regionally or systemically such as through arterial infusion, intraventricular infusion, intratumoral infusion, intraperitoneal infusion, intrapleural infusion, or intravenous infusion. Other methods of delivery may include via a hydrogel or microneedle array.
  • a CAR-T cell as described herein may include a receptor (e.g., an antigen binding molecule) having an amino acid sequence having at least 70%, 75%.
  • a receptor e.g., an antigen binding molecule
  • a B cell e.g., a B lymphocyte
  • a B lymphocyte is a type of blood cell that acts as a center for a population of cells that express clonally diverse cell surface immunoglobulin (Ig) receptors recognizing specific antigenic epitopes (see, e.g., LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood.2008 Sep 1;112(5):1570-80).
  • Ig immunoglobulin
  • B cells include mature B cells, including subtypes thereof, such as, for example and without limitation, transitional B cells, na ⁇ ve B cells, activated B cells, plasma B cells and memory B cells and, in the context of the present invention, precursors thereof, so long as such precursors comprise suitable surface antigens and/or other phenotypic or genotypic characteristics such that they may be subject to regulation as described herein.
  • Activated B cells result from the binding of an antigen to a B cell 37 66H6137.
  • DOCX Attorney Docket No.06527-2502271 receptor such as the binding of an antigen to a B cell receptor of a na ⁇ ve B cell.
  • B cell activation may be done independent of T cells or through T cell dependent pathways.
  • Memory B cells are a long-lived subset of B cells that, after initial activation, circulate throughout the body until they encounter an antigen that binds a B cell receptor on the memory B cell. After the B cell receptor is bound, a memory B cell may differentiate into a plasma cell. All genes and surface markers, as described herein, are broadly- known and do not require further explanation, such as B-cell markers, including, but not limited to CD19, 20, 21, 22, 23, 24, 25, 40, 72, CD79a,b, CD80, and/orCD86 (see, e.g. LeBien TW, et al. Blood.2008 Sep 1;112(5):1570-80).
  • a “CD11c+ B cell” refers to a heterogenous population of B cells that express the CD11c surface antigen. They comprise mainly memory B cells, but may comprise other B cell types (see, e.g., Golinski ML, et al. CD11c+ B Cells Are Mainly Memory Cells, Precursors of Antibody Secreting Cells in Healthy Donors. Front Immunol. 2020 Feb 25;11:32).
  • Age associated B cells (ABCs or ABC cells), may be expanded in the elderly, and also can be associated with various autoimmune diseases, and therefore provide therapeutic targets of the therapies and therapeutic compositions provided herein. ABC cells may be defined as a B cell subset characterized by the CD19 + (see, e.g., Wang K, et al.
  • CD19 a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol.2012 Nov 29;1(1):36), CD21 ⁇ (see, e.g., Suryani S, et al. Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells. Blood. 2010 Jan 21;115(3):519-29), CD11c + (see, e.g., Golinski ML, et al. Front Immunol.2020 Feb 25;11:32), T-bet + (see, e.g., Knox JJ, et al. T-bet+ memory B cells: Generation, function, and fate. Immunol Rev.
  • B cells may be engineered to express an antigen binding molecule as described herein by introducing a construct (for example, including a promoter, coding sequence encoding the antigen binding molecule, a marker for selection, and/or a sequence for CRISPR-mediated knock-in) into the genome of the B cell (e.g., through a vector (such as a lentiviral vector, an AAV vector), electro- or nanoporation, mRNA transfection, and the like known to those of skill in the art) a nucleic acid encoding an antigen binding molecule.
  • a construct for example, including a promoter, coding sequence encoding the antigen binding molecule, a marker for selection, and/or a sequence for CRISPR-mediated knock-in
  • the antigen binding molecule that is expressed has an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 38 66H6137.DOCX Attorney Docket No.06527-2502271 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive.
  • the B-cell may be a B-cell that is autologous to a patient.
  • the engineered B-cell may then be reintroduced to the patient.
  • Antibody–antigen structural complex generation [00172]
  • the COSMIC 2 server https://cosmic-cryoem.org/), a cloud-based computational platform developed by the University of Michigan for structural biology projects and openly accessible to academic researchers, facilitated the generation of an antibody–antigen structural complex.
  • the protein sequence for gp120 Clade C was obtained from the Protein Data Bank (PDB: 7ri1) (https://www.rcsb.org/) and uploaded onto COSMIC 2 , along with the sequence files for HuJ3 and gp120 provided in the Supplementary Material. Subsequently, an AlphaFold2 job was initiated on the server. Since the AlphaFold toolkit on COSMIC 2 does not provide configuration of the number of generated models, the “number of predictions per model” was set to 10 to enhance prediction diversity. [00173] Structure refinement and local docking of antibody–antigen complex [00174] The initial binding pose generated by AlphaFold-Multimer was refined using SnugDock ensemble docking.
  • FlexddG program https://github.com/Kortemme-Lab/flex_ddG_tutorial was applied to perform single point mutation on the antibody interface residues to increase the binding affinity of HuJ3-gp120 complex.
  • the complex we wished to mutate was placed in the “inputs” folder.
  • the antibody chain id was specified in the “chains_to_move.txt” file located in the “inputs” folder.
  • FlexddG only allows users to mutate one residue at a time, so the “run_example_2.py” script was modified to specify the residue chain id and number we wanted to mutate each time. The parameters used to run the program were set as the recommended values.
  • the “analyze_flex_ddG.py” file was used to analyze the output results from “run_example_2.py”, which printed the wild type and mutant interface ⁇ G. ⁇ G score and the ⁇ G score reweighted with the fitted GAM model were calculated and listed in a .csv file through “analyze_flex_ddG.py”.
  • the mutant whose reweighted ⁇ G score was lower than 0 kcal/mol was accepted as the mutation which may increase the binding affinity of the complex.
  • BioLuminate point mutation validation [00179] BioLuminate software was downloaded from the Schrödinger website. The target complex structure was first imported to BioLuminate.
  • the lac promoter is used for transcription initiation followed by an OmpA signal peptide directing VH proteins secretion into periplasm.
  • the six consecutive histidine tag and the following Flag tag were used for protein purification and detection.
  • the pComb3x containing huJ3 was constructed by molecular subcloning to insert the huJ3 gene into the Sfi I linearized pComb3x backbone using the enzymes Sfi I and T4 ligase.
  • the mutants plasmids were made through site- directed mutagenesis at BonOpus. Competent HB2151 Escherichia coli was transformed with these plasmids to express VH nanobodies. Antibodies were purified from E.
  • coli periplasm after polumyxin B treatment followed by Ni-NTA chromatography The molecular size and purity was verified by SDS-PAGE.
  • ELISA was performed to determine quantitative binding of each variant to the HIV-1 envelope glycoprotein.
  • Transiently expressed gp140, a functional homolog of gp120 used to 41 66H6137.DOCX Attorney Docket No.06527-2502271 evaluate HIV-1 binding in vitro, served as the antigen for evaluation. Plates were coated with 50 ng gp140/well at 4°C overnight and subsequently blocked with 3% milk for 1 h at room temperature.
  • Binding activity was detected using 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped after 2 min by TMB stop buffer (ScyTek Laboratories, Logan, UT, USA) to prevent oversaturation and absorbance was read at 450 nm. The experiment was performed in duplicate. After analysis, another ELISA was performed in the same manner as above to validate and compare the gp140 affinity of variant E44R with J3 and HuJ3. [00184] E44R was further validated in the laboratory-developed TZM-bl HIV-1 Phenotyping Assay to measure the susceptibility of HIV-1 (group M, subtype B, isolate BAL) to E44R compared with HuJ3.
  • TZM-bl cells are an indicator cell line that allows quantitative analysis of HIV replication.
  • TZM-bl cells were generated from HeLa cells that stably express large amounts of CD4 and CCR5 and have separately integrated copies of the luciferase and ß-galactosidase genes under control of the HIV-1 promoter. These cells naturally express CXCR4 receptors.
  • TZM-bl cells were plated at 10,000 cells per well overnight. The cells were treated with serial dilutions of HuJ3 antibodies, and infected with a dilution of infectious HIV-1 virus normalized to an output of 140,000 relative light units (RLU) as determined by endpoint dilution.
  • RLU relative light units
  • VHH nanobody J3 (PDB: 7ri1) was selected as the template to model the HuJ3. 42 66H6137.DOCX Attorney Docket No.06527-2502271 [00187] ClusPro was employed to generate the potential binding poses of the HuJ3- gp120 complex. To specify the antibody docking, “Antibody Mode” was chosen. HuJ3 was designated as the receptor and gp120 as the ligand. Paratopes and epitopes were entered into the attraction sections of the receptor and ligand, respectively, to set the docking constraints. The cluster among the top 10 global docking results that closely resembled the binding pose of PDB: 7ri1 was selected as the potential binding pose.
  • Step 2 AlphaFold-Multimer2.3/3.0 generates 3D structures of the antibody, antigen, and their complex, effectively combining the functions of homology modeling and global docking from IsAb1.0.
  • the per-residue confidence metric pLDDT
  • pLDDT per-residue confidence metric
  • the pLDDT scores are below 70, the antibody–antigen complex undergoes further refinement. Otherwise, the complex proceeds to Step 4 for local docking.
  • Step 3 several methods can be used for structural refinement. If the structure with lower pLDDT score has a crystal structure, this crystal structure will be optimized by Rosetta FastRelax to resolve potential clashes and identify energetically favorable conformations.
  • Step 4 SnugDock performs local docking for the antibody and antigen, allowing flexibility of the CDR loops and interfacial side chains. SnugDock refines the potential binding poses provided by AlphaFold-Multimer and outputs the final antibody–antigen complex.
  • Step 5 after obtaining the 3D structure of antibody–antigen complex, alanine scanning is performed to predict possible hotspots on the antibody, facilitating future antibody design.
  • Model_3_Pred_5 contained some structural errors. These errors included a loss of alpha helix between residues LEU369-THR372 and two structural errors in the beta- sheet regions, specifically between residues LYS419-LYS421 and ASP464-GLU466. The pLDTT plot indicated that all these regions had lower pLDTT scores.
  • the SnugDock ensemble docking was employed to perform local docking searches using backbone ensembles, which has been proved to improve docking accuracy.
  • This ensemble docking approach relies on the conformational-selection mechanism for protein docking, utilizing a pre-generated ensemble of protein partners.
  • the ensembles were generated using Rosetta Relax to sample various backbone conformations during docking. In the low-resolution stage, each docking run involves rigid-body translation and rotation around the protein partner, along with backbone swapping from the pre-generated ensemble. This allows for the sampling of diverse backbone conformations.
  • the robust evaluation of successful local docking is the presence of a “docking funnel”, in which the “near-native” result (low I_rmsd) has lower energy (low I_sc) than the non-native result.
  • the “near-native” result (low I_rmsd) has lower energy (low I_sc) than the non-native result.
  • T99R had the lowest ⁇ G value ⁇ 0.979 kcal/mol among the other substitutions.
  • R99 formed two hydrogen bonds with W427 and one with D474, which made the binding between HuJ3 and gp120 more stable (FIG. 48 66H6137.DOCX Attorney Docket No.06527-2502271 4B, panel D).
  • FIG.6 illustrates that the differences between the 3D structures of HuJ3-gp120 complexes generated by IsAb1.0 and IsAb2.0 were minimal. The modeling and docking results from both versions were closely aligned.
  • IsAb2.0 does not require modeled structures to have homologous proteins as template, allowing it to generate 3D structures for novel antibodies and antigens. Additionally, IsAb2.0 does not require pre-existing binding information of the antibody and antigen, making it broadly applicable in various scenarios.
  • FIG. 5 top panel shows that all four mutations predicted by IsAb1.0 exhibited either the same or lower binding affinity compared with HuJ3. In contrast, as discussed previously, one of the five predictions 50 66H6137.DOCX Attorney Docket No.06527-2502271 from IsAb2.0 was shown to increase the HuJ3-gp120 binding affinity, indicating that IsAb2.0 achieves higher prediction accuracy than IsAb1.0.
  • IsAb2.0 an advanced antibody design protocol, by integrating state-of-the-art AI-based and physical methods. IsAb2.0 can be utilized for the design of humanized antibodies and nanobodies, enabling the construction of more accurate models of antibody–antigen complexes without the need for template and additional binding information, using AlphaFold-Multimer. Furthermore, IsAb2.0 employs more accurate method FlexddG for predicting mutations.
  • AlphaFold-Multimer often fails to accurately model secondary structures, which can affect local docking accuracy. Users can evaluate the quality of structures modeled by AlphaFold-Multimer based on the pLDDT scores of residues. According to the AlphaFold official document, regions with pLDDT scores lower than 70 have low confidence and should be treated cautiously. We suggest that if pLDDT scores of all regions, or the interaction critical regions, are higher than 70, users can directly input complex modeled by AlphaFold-Multer into SnugDock. In our case, some secondary structures of both the antibody and antigen were not accurately predicted. Hence, structure refinement is necessary to achieve higher structural accuracy for the antibody or antigen.
  • the best way to perform structure refinement is to replace the structures of both the antibody and antigen modeled by AlphaFold- Multimer with high-resolution crystal structures. If the crystal structures are unavailable, users can generate accurate 3D structures by some powerful modeling program, e.g. SWISS-MODEL. 52 66H6137.DOCX Attorney Docket No.06527-2502271 [00210]
  • SWISS-MODEL. 52 66H6137.DOCX Attorney Docket No.06527-2502271 [00210]
  • a standard, user-friendly antibody design tool could significantly improve and accelerate the development of antibody therapies across several fields, including the treatment of cancers, viral infections, and addiction.

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Abstract

OF THE INVENTION Provided herein is a humanized J3 derivative antigen binding molecule and uses thereof.

Description

Attorney Docket No.06527-2502271 HUMANIZATION AND AFFINITY MATURATION OF THE CD4-MIMICKING LLAMA NANOBODY J3 CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Patent Application No. 63/682,415, filed August 13, 2024, the disclosure of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under grant number AI164556 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCING LISTING [0003] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 2502271.xml. The size of the XML file is 20,655 bytes and the XML file was created on August 12, 2025. BACKGROUND OF THE INVENTION Field of the Invention [0004] Provided herein are compositions and methods of using the same for treatment of various conditions, including those caused by human immunodeficiency virus (HIV). Description of Related Art [0005] Human Immunodeficiency Virus (HIV) remains a chief global health concern in the 21st century. Current treatments for HIV include a variety of antiretroviral therapies (ART) aimed at decreasing the patent’s viral load to alleviate symptoms and prevent further transmission of the disease. However, the relatively high failure rate and side effects of long-term ART necessitates novel treatments that can inhibit HIV- 1 and prevent viral escape in infected individuals. Antibody therapeutics are one such therapeutic modality that may be capable of filling this gap in treatment. An antibody’s high affinity for its particular antigen allows these treatments to effectively target specific cells and reduce potential off-target effects of the therapy. 1 66H6137.DOCX Attorney Docket No.06527-2502271 [0006] Mulyiple broadly neutralizing antibodies (bnAbs) targeting different HIV-1 envelope glycoproteins (gp160) sites have been isolated and characterized. Some of them have shown efficacy in animal studies and early human trials for both treatment and prevention. One challenge for bnAb is the viral escape, which necessitates development of combination therapy of bnAbs. Thus, there is a need in the art for new treatments. SUMMARY OF THE INVENTION [0007] Provided herein is a humanized J3 derivative antigen binding molecule. [0008] Also provided herein is a humanized antigen binding molecule including a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0009] Also provided herein is a method of binding or neutralizing a human immune deficiency virus (HIV) particle, including contacting the HIV particle with a humanized J3 derivative antigen binding molecule. [0010] Also provided herein is a method of treating a patient infected with HIV including administering to the patient a humanized J3 derivative antigen binding molecule to the patient in an amount effective to treat the infection. [0011] Also provided herein is a composition including a humanized J3 derivative antigen binding molecule in a pharmaceutically-acceptable excipient, vehicle, or carrier. [0012] Also provided herein is a CAR-T cell expressing an antigen binding molecule having the amino acid sequence of SEQ ID NO: 14. [0013] Also provided herein is a CAR-T cell expressing an antigen binding molecule having the amino acid sequence of SEQ ID NO: 3. [0014] Also provided herein is a CAR-T cell expressing an antigen binding molecule having the amino acid sequence of SEQ ID NO: 9. [0015] Also provided herein is a B-cell engineered to express an antigen binding molecule having the amino acid sequence of SEQ ID NO: 14. [0016] Also provided herein is a B-cell engineered to express an antigen binding molecule having the amino acid sequence of SEQ ID NO: 3. 2 66H6137.DOCX Attorney Docket No.06527-2502271 [0017] Also provided herein is a B-cell engineered to express an antigen binding molecule having the amino acid sequence of SEQ ID NO: 9. [0018] Further non-limiting embodiments are set forth in the following numbered clauses: [0019] 1. A humanized J3 derivative antigen binding molecule. [0020] 2. The humanized antigen binding molecule of clause 1, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. [0021] 3. The antigen binding molecule of clause 1 or clause 2, wherein the amino acid sequence comprises SEQ ID NO: 14. [0022] 4. The antigen binding molecule of any of clauses 1-3, wherein the amino acid sequence consists of SEQ ID NO: 14. [0023] 5. The humanized antigen binding molecule of any of clauses 1-4, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. [0024] 6. The antigen binding molecule of any of clauses 1-5, wherein the amino acid sequence comprises SEQ ID NO: 3. [0025] 7. The antigen binding molecule of any of clauses 1-6, wherein the amino acid sequence consists of SEQ ID NO: 3. [0026] 8. The humanized antigen binding molecule of any of clauses 1-7, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [0027] 9. The antigen binding molecule of any of clauses 1-8, wherein the amino acid sequence comprises SEQ ID NO: 9. [0028] 10. The antigen binding molecule of any of clauses 1-9, wherein the amino acid sequence consists of SEQ ID NO: 9. [0029] 11. A humanized antigen binding molecule comprising a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 3 66H6137.DOCX Attorney Docket No.06527-2502271 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0030] 12. A method of binding or neutralizing a human immune deficiency virus (HIV) particle, comprising contacting the HIV particle with a humanized J3 derivative antigen binding molecule. [0031] 13. The method of clause 12, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. [0032] 14. The method of clause 12 or clause 13, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. [0033] 15. The method of any of clauses 12-14, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. [0034] 16. The method of any of clauses 12-15, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. [0035] 17. The method of any of clauses 12-16, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. [0036] 18. The method of any of clauses 12-17, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. [0037] 19. The method of any of clauses 12-18, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [0038] 20. The method of any of clauses 12-19, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. [0039] 21. The method of any of clauses 12-20, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. [0040] 22. The method of any of clauses 12-21, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID 4 66H6137.DOCX Attorney Docket No.06527-2502271 NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0041] 23. The method of any of clauses 12-22, wherein the HIV particle is an HIV1 particle. [0042] 24. A method of treating a patient infected with HIV comprising administering to the patient a humanized J3 derivative antigen binding molecule to the patient in an amount effective to treat the infection. [0043] 25. The method of clause 24, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. [0044] 26. The method of clause 24 or clause 25, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. [0045] 27. The method of any of clauses 24-26, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. [0046] 28. The method of any of clauses 24-27, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. [0047] 29. The method of any of clauses 24-28, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. [0048] 30. The method of any of clauses 24-29, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. [0049] 31. The method of any of clauses 24-30, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [0050] 32. The method of any of clauses 24-31, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. [0051] 33. The method of any of clauses 24-32, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. [0052] 34. The method any of clauses 24-33, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, 5 66H6137.DOCX Attorney Docket No.06527-2502271 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0053] 35. The method of any of clauses 24-34, wherein the HIV is HIV1. [0054] 36. A composition comprising a humanized J3 derivative antigen binding molecule in a pharmaceutically-acceptable excipient, vehicle, or carrier. [0055] 37. The composition of clause 36, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. [0056] 38. The composition of clause 36 or clause 37, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. [0057] 39. The composition of any of clauses 36-38, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. [0058] 40. The composition of any of clauses 36-39, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. [0059] 41. The composition of any of clauses 36-40, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. [0060] 42. The composition of any of clauses 36-41, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. [0061] 43. The composition of any of clauses 36-42, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [0062] 44. The composition of any of clauses 36-43, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. [0063] 45. The composition of any of clauses 36-44, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. [0064] 46. The composition of any of clauses 36-45, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID 6 66H6137.DOCX Attorney Docket No.06527-2502271 NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. [0065] 47. The composition of any of clauses 36-46, formulated for parental administration. [0066] 48. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 14. [0067] 49. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 3. [0068] 50. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 9. [0069] 51. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 14. [0070] 52. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 3. [0071] 53. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 9. BRIEF DESCRIPTION OF THE DRAWINGS [0072] FIG. 1 shows the workflow of the IsAb2.0; users are required to input the sequences of antibody and antigen into IsAb2.0 protocol; AlphaFold-Multimer 2.3/3.0 generates 3D structure of antibody–antigen complex based on their sequences; if the prediction quality (pLDDT) of complex is below 70, the structure undergoes refinement; SnugDock is then used to refine the antibody–antigen complex, followed by alanine scanning and FlexddG for hotspot identification and antibody optimization. [0073] FIG. 2 shows comparison between HuJ3-gp120 and J3-gp120 3D structures. We overlapped the HuJ3-gp120 complex generated by IsAb2.0 with the J3-gp120 crystal complex. The general binding poses of HuJ3 and J3 with gp120 are highly similar, especially their CDR loops. [0074] FIG. 3 shows comparison of hydrogen bonds between HuJ3 and J3 with gp120. Yellow dash line is hydrogen bond. (A) and (B) are the hydrogen bonds on HuJ3-gp120. (C) and (D) are the hydrogen bonds on J3-gp120. Most of the residue pairs forming hydrogen bonds are the same between HuJ3-gp120 and J3-gp120 complexes. Predicted epitopes of gp120 (D368, N425, M426, and V430) form hydrogen bonds with HuJ3. 7 66H6137.DOCX Attorney Docket No.06527-2502271 [0075] FIGS. 4A-4C show comparison of hydrogen bonds between HuJ3 and designed HuJ3 with gp120. The yellow dashed line is the hydrogen bond. (A) E44R mutation, R44 enhances electrostatic interactions favorably. (B) V54E mutation, E54 forms two hydrogen bonds with R432 compared with V54. (C) S98W mutation, W98 increases the electrostatic property of the HuJ3-gp120 complex. (D) T99R mutation, T99 forms a hydrogen bond with M426, and R99 forms two hydrogen bonds with W427 and one with D474. (E) S102R mutation, R102 forms hydrogen bonds with P470 and I371 on gp120, respectively. [0076] FIG.5 shows experimental validation of designed HuJ3 variants. (A) Binding affinity of HuJ3 variants predicted by IsAb1.0 for HIV-1 Bal gp140. (B) Binding affinity of HuJ3 variants predicted by IsAb2.0 for HIV-1 Bal gp140. (C) Percent neutralization of HIV-1 Bal by HuJ3 variants predicted by IsAb2.0 [0077] FIG.6 shows alignment of the HuJ3-gp120 complexes generated by IsAb1.0 and IsAb2.0. [0078] FIG. 7 shows sequences of various nanobodies, including HuJ3 (SEQ ID NO: 1) and NanoJ3 (SEQ ID NO: 2), according to non-limiting embodiments disclosed herein. [0079] FIG.8 shows sequences of various nanobodies, including full-length HuJ3 (SEQ ID NO: 14) and 4E2 (SEQ ID NO: 3), according to non-limiting embodiments disclosed herein. [0080] FIG. 9 shows sequences of various nanobodies, including K97T (SEQ ID NO: 4), N71E (SEQ ID NO: 5), H56Y (SEQ ID NO: 6), full-length E44R (SEQ ID NO: 7), full-length HuJ3 (SEQ ID NO: 14), and N30T (SEQ ID NO: 8), according to non- limiting embodiments disclosed herein. [0081] FIG. 10 shows sequences of various nanobodies, including full-length NanoJ3 (SEQ ID NO: 15), 4E2 (SEQ ID NO: 3), full-length HuJ3 (SEQ ID NO: 14), and G12 (SEQ ID NO: 9), according to non-limiting embodiments disclosed herein. [0082] FIG.11 shows the sequence alignment of 5 designed mutations by IsAb2.0, including E44R (SEQ ID NO: 16), V54E (SEQ ID NO: 10), S98W (SEQ ID NO: 11), T99R (SEQ ID NO: 12), and S102R (SEQ ID NO: 13). The amino acids marked as black are the amino acids being mutated compared with HuJ3 (SEQ ID NO: 1). 8 66H6137.DOCX Attorney Docket No.06527-2502271 DESCRIPTION OF THE INVENTION [0083] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein "a" and "an" refer to one or more. [0084] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by". As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps. [0085] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise indicated, polymer molecular weight is expressed as number-average molecular weight (Mn). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. [0086] The term “contacting” refers to placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell. In other examples, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample. [0087] An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in the cell of the organism in which the 9 66H6137.DOCX Attorney Docket No.06527-2502271 component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified”, thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. A preparation may be purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation. [0088] A nucleic acid molecule (a nucleic acid) refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. [0089] A first nucleic acid is said to be operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. Generally, operably linked DNA sequences are contiguous (e.g., in cis) and, where the sequences act to join two protein coding regions, in the same reading frame (e.g., open reading frame or ORF), for example to produce a fusion protein. Operably linked nucleic acids include a first nucleic acid contiguous with the 5′ or 3′ end of a second nucleic acid. In other examples, a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid. [0090] A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species). For example, a nucleic acid 10 66H6137.DOCX Attorney Docket No.06527-2502271 sequence can be optimized for expression in yeast cells. Codon optimization does not alter the amino acid sequence of the encoded protein. [0091] A conservative substitution is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, an antigen binding molecule or antibody polypeptide sequence may include one or more conservative substitutions (for example 1-10, 2-5, or 10-20, or no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) yet retains the affinity or avidity of a given antigen binding molecule such as those described herein for binding to, among other possibilities, human immunodeficiency virus (HIV). A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Methods are provided herein to ascertain proper expression of any sequence. [0092] A polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide”, “peptide”, or “protein” as used herein are intended to encompass any amino acid sequence and include proteins and modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are synthetically produced such as by recombinant or chemical synthesis methods. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide. [0093] Conservative amino acid substitutions are those substitutions that, when made, least or minimally interfere with the properties of the original protein, that is, in the context of the end-use, the structure and function of the protein is conserved and not significantly changed by such substitutions, and may be identified by use of matrices, such as the BLOSUM series of matrices, and other matrices. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a 11 66H6137.DOCX Attorney Docket No.06527-2502271 hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine. In terms of antibody structure, conservative substitutions may be relatively freely made to framework amino acids and constant region amino acids, such as to humanize an antigen binding molecule. [0094] As used herein, the term “epitope” refers to a physical structure or moiety of a molecule that interacts with an antibody or antibody binding reagent. In terms of proteins or polypeptides, the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications, can define an epitope. For example, HIV protein and protein fragments, including isoforms and post-transcriptionally-modified polypeptides for use in the immunodetection methods, devices, and kits may be produced in mammalian cells, such as HEK293 cells, to produce a protein with mammalian post-translational modifications. Portions of a natural protein can contain an epitope present in the complete natural protein and typically react to antibodies raised to the natural protein. [0095] A recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein. [0096] “Sequence identity” refers to the similarity between nucleic acid or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity may be measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, isoforms, or variants of a polypeptide often possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well- known in the art. Various programs and alignment algorithms are described in the art 12 66H6137.DOCX Attorney Docket No.06527-2502271 (see, e.g., Chao J, et al. Developments in Algorithms for Sequence Alignment: A Review. Biomolecules.2022 Apr 6;12(4):546). [0097] Once aligned, the number of matches may be determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity may be determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value may be rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. [0098] Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants may typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. [0099] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences may be entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters 13 66H6137.DOCX Attorney Docket No.06527-2502271 may be used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example and without limitation, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithm of Needleman & Wunsch, by the search for similarity method of Pearson & Lipman, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by manual alignment and visual inspection. One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle. Using PILEUP, a reference sequence may be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package (see, e.g., Chao J, et al. Biomolecules.2022 Apr 6;12(4):546). [00100] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program may be used for nucleotide sequences. The BLASTP program may be used for amino acid sequences. [00101] As used herein, reference to “at least 70% identity” (or similar language) may refer to “at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. As used herein, reference to “at least 90% identity” (or similar language) may refer to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. [00102] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide strands that are typically in antiparallel orientation. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. In RNA as opposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences 14 66H6137.DOCX Attorney Docket No.06527-2502271 comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1X SSC (saline sodium citrate) to 10X SSC, where 1X SSC is 0.15M NaCl and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by the nucleobase content of the strands, the presence of mismatches, the length of complementary sequences, salt concentration, temperature, with the melting temperature (Tm) lowering with shorter complementary sequences, increased mismatches, and increased stringency. Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other. [00103] A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. [00104] By “expression” or “gene expression,” it is meant the overall flow of information from a gene. A “gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as an ncRNA that has a function. A “gene” is a functional genetic unit for producing its gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and generally comprising: a transcriptional control sequence, such as a promoter and other cis- acting elements, such as transcriptional response elements (TREs) and/or enhancers; an expressed sequence that typically encodes a protein (referred to as an open- reading frame or ORF) or functional/structural RNA; and a polyadenylation sequence). A gene produces a gene product (typically a protein, optionally post-translationally modified, or a functional/structural RNA) when transcribed. By “expression of genes under transcriptional control of,” or alternately “subject to control by” a designated sequence such as a promotor, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene. A gene that is “under transcriptional control” of an inducible promotor or transcription control element, is a gene that is transcribed at detectably different levels 15 66H6137.DOCX Attorney Docket No.06527-2502271 in the presence of a transcription factor, e.g., in specific cell types or conditions. A “gene for expression of” a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment, that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression. In the case of a constitutive promoter “suitable conditions” means that the gene typically need only be introduced into a host cell. In the case of an inducible promoter, “suitable conditions” means when factors that regulate transcription, such as DNA-binding proteins, are present or absent, for example, an amount of the respective inducer is available to the expression system (e.g., cell), or factors causing suppression of a gene are unavailable or displaced - effective to cause expression of the gene. [00105] In further detail, transcription is the process by which the DNA gene sequence is transcribed into RNA. The steps include transcript initiation, transcript elongation, and transcript termination. The molecular machinery of transcription includes but is not limited to: RNA polymerase, general transcription factors, enhancers, and promoter DNA, and RNA transcript. Transcription factors (TFs) are proteins that control the rate of transcription of genetic information from DNA to RNA, by binding to a specific DNA sequence (e.g., the promoter region). The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. The promoter region of a gene is a region of DNA that initiates transcription of that particular gene. Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100–1000 base pairs long. Additional sequences and non-coding elements can affect transcription rates. If the cell has a nucleus (eukaryotes), the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation refers to the addition of a poly(A) tail to a messenger RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide. In eukaryotes, the 5’ cap (cap-0), found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage. During RNA splicing, pre-mRNA is 16 66H6137.DOCX Attorney Docket No.06527-2502271 edited. Specifically, during this process introns are removed, and exons are joined together. The resultant product is known as mature mRNA. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex. [00106] Gene expression involves various steps, including transcription, post- transcriptional RNA modification, translation, and post-translational modification of a protein. Expression of a gene may also include reduction of the total amount of the protein product, such as by cleavage, sequestration, binding, or other means of decreasing the function or amount of a protein product. [00107] Nucleic acids and vectors encoding the described fusion proteins may be provided. In some non-limiting examples, disclosed is a recombinant vector, such as a yeast plasmid, that expresses the disclosed fusion proteins. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence, but which encode the same protein sequence due to codon degeneracy. In some embodiments, the polynucleotide is codon-optimized for expression in mammalian cells. [00108] Exemplary nucleic acids may be prepared by cloning techniques, e.g., as are broadly-known and implemented either commercially, or in the art. Multiple textbooks and reference manuals describe and provide examples of useful and appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through such techniques are known. Commercial and public product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), Addgene, and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources. [00109] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill. 17 66H6137.DOCX Attorney Docket No.06527-2502271 [00110] Provided herein are antigen binding molecules, e.g., antibody compounds comprising an antibody domain targeting a protein, for example an HIV protein, for example an HIV gp120 protein (including, without limitation, recombinant proteins) and epitopes, and methods of use of those compounds. The term “antigen binding molecule”, for ease of reference and unless otherwise specified, refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan A, Marcatili P, Tramontano A. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 Aug;278(16):2858-66, and US Patent No. 11,578,428 B2, and U.S. Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies). As such, the antigen binding molecule operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen. The term “VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be “humanized” to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE. [00111] An antigen binding molecule or complexes thereof may be, for example and without limitation, a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab′, Fv fragments, single chain Fv (scFv) fragments, dsFv, Fab1 fragments, F(ab′)2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific 18 66H6137.DOCX Attorney Docket No.06527-2502271 T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition. [00112] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., “Protein engineering of single-chain Fv analogs and fusion proteins, ”Methods in Enzymology, vol.203, pp.46–88, 1991; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; Gąciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci.2020 Aug 31;21(17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol.2019 Nov;103(21-22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71; see, also, www.kbdna.com/cloning-scfv [00113] The antigen binding molecules described herein, comprise, at their core paratopes formed from VL and VH polypeptides, that are defined by three CDR’s (typically loops), CDR1, CDR2, and CDR3, which for VH peptides may be termed HCDR1, HCDR2, and HCDR3, respectively, and which for VL peptides may be termed LCDR1, LCDR2, and LCDR3, respectively, each of which are flanked by, and separated by framework (e.g., joining or scaffold) amino acid sequences that space apart and support the CDRs, and which may differ from antibody-to-antibody, and which may be “humanized” to minimize antigenicity when administered to a human 19 66H6137.DOCX Attorney Docket No.06527-2502271 patient. In nature, HCDR3 and LCDR3 are typically the most variable of the CDRs, contributing significantly to antibody specificity. Various methods may be used to identify the precise limits of each CDR, but the sequences provided herein can be evaluated by any suitable method to determine the CDRs. [00114] Exemplary sequences of antibody heavy and light chains are provided in the attached sequence listing, which is incorporated herein by reference in its entirety. Antibody constant and variable regions, including CDR sequences and framework sequences can be readily ascertained from the sequences provided in in the attached sequence listing. Reference to a CDR herein may refer to a Kabat CDR numbering scheme or any other applicable CDR numbering scheme, including but not limited to Chothia, Martin (enhanced Chothia), Gelfand, IMGT, Honneger, or any other numbering scheme (see, e.g., Dondelinger M, et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol. 2018 Oct 16;9:2278). For example and without limitation, antibody sequence annotation, including definition of CDR sequences may be conducted using tools described and provided in abYsis (abysis.com), or Abnum (www.bioinf.org.uk/abs/abnum/). Other methods of CDR identification are known in the art (see, e.g., Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4; Adolf- Bryfogle J, Xu Q, North B, Lehmann A, Dunbrack RL Jr. PyIgClassify: a database of antibody CDR structural classifications. Nucleic Acids Res. 2015 Jan;43(Database issue):D432-8), and assorted online tools and applications as are broadly-available. As such, an amino acid sequence of a VH or VL may be provided or determined, comprising CDRs, and one of ordinary skill can determine the precise metes and bounds of CDRs within that antibody sequence without undue experimentation. Framework sequences may be optimized (see, e.g., Gopal R, Fitzpatrick E, Pentakota N, Jayaraman A, Tharakaraman K, Capila I. Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti- SARS-CoV-2 Antibody. Viruses.2022 Nov 30;14(12):2694) and/or humanized based on knowledge of amino acid sequences of the CDRs, e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of antibodies described herein. [00115] Antibodies may be produced by any effective method, such as by hybridoma or it may be recombinantly or synthetically produced. In the context of the 20 66H6137.DOCX Attorney Docket No.06527-2502271 present disclosure and for ease of reference, “antibodies” or “antibody” may refer to both natural antibodies as well as protein antibody analogs, antibody fragments, and derivatives, any of which comprising VL and/or VH sequences and/or CDRs (e.g., all three CDRs of any VH or VL region, defining a paratope as described herein) according to any example, aspect, or embodiment described herein. The antibody or antibodies may be synthetic, in that they do not comprise a naturally-occurring sequence, such as certain antigen binding molecules, including engineered versions and derivatives thereof, such as scFv versions thereof, humanized versions thereof, BiTEs, and/or sequence derivatives thereof, including without limitation, proteins comprising the CDRs (e.g., one or more, or all three CDRs) of the antibodies provided herein. [00116] Nanobodies, which may be referred to a VHH antibodies or single-domain antibodies, may be constructed using CDR sequences, such as CDRs of the antibodies described herein. Nanobodies may be created by grafting of the complementarity determining regions (CDRs) from already existing, non-camelid antibodies to VHH frameworks, followed by affinity maturation using synthetic phage libraries (see, e.g., Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two- Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci.2018 Nov 2;19(11):3444). A VH, alone, may be capable of defining an antigen-binding site (e.g., paratope) with sufficient strength to be pharmacologically-useful, and can be referred to as a nanobody (see, e.g., Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 2009 Aug;198(3):157-74, providing structure and sequences of various nanobodies and Bever CS, Dong JX, Vasylieva N, Barnych B, Cui Y, Xu ZL, Hammock BD, Gee SJ. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016 Sep;408(22):5985- 6002). Single-domain antigen binding molecules originally were camelid antibodies, which naturally comprise only heavy chains (see, e.g., Mitchell LS, Colwell LJ. Comparative analysis of nanobody sequence and structure data. Proteins. 2018 Jul;86(7):697-706). More recently other single-chain antigen-binding molecules have been developed. Construction and humanization of single-domain antigen binding molecules is broadly-known (see, e.g., Valdés-Tresanco MS, Molina-Zapata A, Pose 21 66H6137.DOCX Attorney Docket No.06527-2502271 AG, Moreno E. Structural Insights into the Design of Synthetic Nanobody Libraries. Molecules.2022 Mar 28;27(7):2198; Wu Y, Jiang S, Ying T. Single-Domain Antibodies As Therapeutics against Human Viral Diseases. Front Immunol.2017 Dec 13;8:1802; Hoey RJ, Eom H, Horn JR. Structure and development of single domain antibodies as modules for therapeutics and diagnostics. Exp Biol Med (Maywood). 2019 Dec;244(17):1568-1576; Rossotti MA, Bélanger K, Henry KA, Tanha J. Immunogenicity and humanization of single-domain antibodies. FEBS J. 2022 Jul;289(14):4304-4327; and Khodabakhsh F, Behdani M, Rami A, Kazemi-Lomedasht F. Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies. Int Rev Immunol. 2018;37(6):316-322). Multimerization methods are broadly-known, too (see, e.g., Miller A, Carr S, Rabbitts T, Ali H. Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats. MAbs. 2020 Jan-Dec;12(1):1752529), for example to produce bi-specific antibody binding molecules, such as bi-specific T-cell engagers (e.g., BiTEs), discussed in further detail, below. Nanobody construction has been commercialized, e.g. in Crescendo Biologics’ Humabody platform (see, Teng Y, et al., Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse. N Biotechnol. 2020 Mar 25;55:65-76, US 11,547,099 B2, and WO 2016/062988 for exemplary constructs, transgenic mice, and methods for producing VH nanobodies). The term “antigen-binding molecule” is used interchangeably with “antibody” and “nanobody” herein. [00117] An antibody-drug conjugate (ADC) may be provided. An antigen binding molecule according to any aspect, embodiment, or example provided described herein may be linked to a payload (e.g., a cargo or warhead) that causes a desired physiological effect, such as killing a cell expressing a binding partner to the antigen binding molecule. The antigen binding molecule, e.g. an antibody, can be effectively covalently-linked to other moieties, for example by their Fc sequences yet retain significant antigen-binding capacity. ADCs comprise an antigen binding moiety (a linked antigen-binding molecule), a linker that may be cleavable or non-cleavable, and the payload moiety. A “moiety” is a chemical group or entity, often functional, that forms part of a larger molecule. Linkers may be used to join a payload moiety to the antigen binding molecule, and choice of linkers can depend on how the APC is handled by the cell, and how the payload becomes effective on processing by a cell, or by release due to chemical lability of the linker. Non-cleavable linkers include, without limitation, 22 66H6137.DOCX Attorney Docket No.06527-2502271 alkyl moieties, and thioether moieties (e.g., Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC)). Cleavable or labile linkers may include, for example and without limitation, acid-labile linkers (hydrolysable in lysosomes or endosomes), Lysosomal protease–sensitive linkers (e.g., peptide-based linkers), β-glucuronide linkers, and glutathione-sensitive disulfide linkers, with examples including, without limitation: ester-, hydrazone-, Valine-citrulline (v-c)-, Valine-alanine (v-a)-, and phenylalanine-lysine (p-l)-containing linkers. (see, e.g., Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res.2020 Jan;18(1):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads). Examples of payloads include, without limitation: microtubule-disrupting agents, such as auristatin, maytansinoids, eribulin (e.g., eribulin mesylate), tubulysins, cryptophycins, and EG5 inhibitors; DNA-damaging agents, such as, without limitation calicheamicin, duocarmycins, doxorubicin, enediyne, topoisomerase I inhibitors, and Pyrrolo[2,1- c][1,4] benzodiazepines; RNA-targeting payloads, such as thailanstatins and amatoxins; immune payloads, such as Toll-like receptor agonists, STING agonists, glucocorticoid receptor modulators; and other payloads, such as Bcl-xL inhibitors, NAMPT inhibitors, and proteasome inhibitors such as carmaphycins. Design and optimization considerations for production of ADCs are provided in Khongorzul P, et al. (Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020 Jan;18(1):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads, and see, e.g., Gogia P, Ashraf H, Bhasin S, Xu Y. Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of Evidence. Cancers (Basel).2023 Jul 30;15(15):3886; Baah S, Laws M, Rahman KM. Antibody-Drug Conjugates-A Tutorial Review. Molecules. 2021 May 15;26(10):2943; and Wang Z, Li H, Gou L, Li W, Wang Y. Antibody-drug conjugates: Recent advances in payloads. Acta Pharm Sin B. 2023 Oct;13(10):4025-4059). ADCs with multiple payloads, PROTAC-guided ADCs, ADCs with peptide-drug-conjugates, and ADCs with photo-reactive payloads also may be produced. As such a person of ordinary skill can produce, based on the teachings herein and without undue experimentation, an ADC comprising an antigen binding molecule as described herein linked to a cytotoxic payload via a chemical linker. [00118] While specific types of antigen binding molecules are described specifically herein, any antigen binding molecule comprising CDR sequences as described herein 23 66H6137.DOCX Attorney Docket No.06527-2502271 for binding an HIV protein is contemplated. Such antigen binding molecules may be evaluated and used in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Müller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes. Front Mol Biosci. 2020 May 27;7:98), single-molecule array (e.g., SIMOA), surface plasmon resonance (SPR), oblique- incidence reflectivity difference (OI-RD) binding affinity, or cell binding assay, are contemplated. Antigen binding molecules with high binding affinities may bind to their corresponding antigen with a KD of 1 µM or less, 500 nM or less, 100nM or less, 75nM or less, 50nM or less, or 25 nM or less. Such antigen binding molecules may find use in therapies for conditions related to (e.g., responsive to) MET agonists, and for diagnostic purposes. [00119] By “target-specific” or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S. Food and Drug Agency (FDA), in aspects, in the context of administering a reagent as described herein to a patient. [00120] As used herein, the “treatment” or “treating” of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure (e.g., an antigen binding molecule or antibody as described herein) with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for a condition associated with a viral protein, including an HIV protein, and also includes monitoring the patient by any useful method, including by use of an antigen binding molecule described herein. In non-limiting embodiments, the condition is HIV and/or AIDS. [00121] A "therapeutically effective amount" refers to an amount of a drug product or active agent effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An “amount effective” for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point. The “amount effective” is preferably safe 24 66H6137.DOCX Attorney Docket No.06527-2502271 - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. Examples of an effective amount of an active agent compounded in a delivery vehicle includes from 100 CAR-T cells per kilogram of body weight to 1X108 CAR-T cells per kilogram of body weight, including any increment therebetween, 1X104 CAR-T cells per kilogram of body weight to 1X107 CAR-T cells per kilogram of body weight, such as ranging from 103, from 104, or from 105 CAR-T cells to 106, to 107, to 108, to 109, to 1010, to 1011, or to 1012 CAR-T cells per dose, including any increment therebetween, such as approximately 103, 104, 105, 105, 106, 107, 108, 109, 1010, or 1011 CAR-T cells per dose. [00122] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate compositions, such as parenteral or inhaled compositions, in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. [00123] An “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose, for example, an amount of an active agent (antigen 25 66H6137.DOCX Attorney Docket No.06527-2502271 binding molecule or antibody) described herein effective to treat a condition described herein. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product. Examples of an effective amount of an active agent compounded in a delivery vehicle includes from 1 µg/ml (micrograms per milliliter) to 100 mg/ml of solution, including any increment therebetween, such as from 1 µg/ml to 1 mg/ml (milligram/milliliter), all values and subranges therebetween inclusive. [00124] Drug products, or pharmaceutical compositions comprising an active agent (e.g., drug), may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s). As used herein, a “pharmaceutically acceptable excipient”, “carrier”, or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohol’s such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the active agent. In certain aspects, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art. The preferred form may depend on the intended mode of administration and therapeutic application, which will in turn dictate the types of carriers/excipients. Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms. [00125] Pharmaceutical formulations adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or 26 66H6137.DOCX Attorney Docket No.06527-2502271 the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts. [00126] Pharmaceutical formulations adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion bags, or the like, as are broadly-known to those of ordinary skill. Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution. [00127] Pharmaceutical formulations adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery. [00128] Pharmaceutical formulations adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. [00129] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. For example, sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization. An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. [00130] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. [00131] The therapeutic agents described herein can be administered by any effective route. Examples of delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, 27 66H6137.DOCX Attorney Docket No.06527-2502271 intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances. Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a composition comprising the therapeutic agent useful for treatment of graft rejection as described herein. [00132] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate therapeutic agents in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of sensitivity in individuals. [00133] Provided herein are antigen binding molecules, such as nanobodies, for binding antigens of interest while minimizing viral escape. The binding molecules described herein can, in non-limiting embodiments, mimic CD4, for example human CD4, thus allowing for effective neutralization of viruses, such as human immunodeficiency virus (HIV), that utilize CD4 as an entry route to a cell. [00134] Accordingly, in non-limiting embodiments, an antigen binding molecule as described herein may be a J3 derivative antigen binding molecule. A J3 antigen binding molecule may be, in non-limiting embodiments, a nanobody obtained from a camelid (e.g., Camelus dromedarius). In non-limiting embodiments, the nanobody is obtained from a llama (e.g., Lama glama). In non-limiting embodiments, the J3 derivative antigen binding molecule may be a humanized binding molecule (e.g., a humanized nanobody). [00135] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 28 66H6137.DOCX Attorney Docket No.06527-2502271 95%, 99%, and/or 100% sequence identity to one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20, all values and subranges therebetween inclusive. [00136] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00137] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 2, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00138] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 3, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00139] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 4, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00140] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 5, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00141] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 6, all values and subranges 29 66H6137.DOCX Attorney Docket No.06527-2502271 therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00142] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 7, all values and subranges therebetween inclusive. [00143] In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 8, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00144] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 9, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00145] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 10, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 11, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00146] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 12, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. 30 66H6137.DOCX Attorney Docket No.06527-2502271 [00147] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 13, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00148] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 14, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00149] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 15, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00150] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 16, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00151] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 17, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00152] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 18, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00153] In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 19, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may 31 66H6137.DOCX Attorney Docket No.06527-2502271 be varied, so long as the CD4 binding site remains intact and/or able to bind CD4.In non-limiting embodiments, an antigen binding molecule as described herein may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 20, all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00154] Also provided herein is a humanized antigen binding molecule that includes a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), or a sequence having at least 70%, 75%. 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to any of the foregoing, all values and subranges therebetween inclusive. [00155] Antigen binding molecules, including humanized antigen binding molecules, as described herein may be used for any purpose, for example, for binding assays (e.g., detection and/or diagnoses) such as immunoprecipitation assays and/or blotting assays (e.g,, Western Blot) as are known in the art. In non-limiting embodiments, an antigen binding molecule as described herein may be used in prophylactically and/or in treatments, for example to bind and/or neutralize a microorganism, such as a virus (e.g., a virus particle). In non-limiting embodiments, an antigen binding molecule as described herein may be used to neutralize HIV. Such a method may include contacting an HIV particle with a nanobody as described herein. In non-limiting embodiments, the HIV particle is an HIV-1 particle. In non-limiting embodiments, the HIV particle is an HIV-2 particle. In non-limiting embodiments, the virus is any virus that may enter and/or infect cells via a CD4-mediated pathway. As described herein, an antigen binding molecule may be formulated for delivery by any suitable route, such as intramuscular, subcutaneous, intraperitoneal, transdermal, and/or the like, with any suitable carriers, vehicles, adjuvants, and/or excipients, and delivered in any suitable concentration/amount for as many doses and/or for as long as is required to provide prophylaxis and/or treatment (e.g., a therapeutically-effective amount). In non-limiting embodiments, antigen binding molecule may be administered 32 66H6137.DOCX Attorney Docket No.06527-2502271 to a patient in an amount of about 50 to about 100 mg/kg, all values and subranges therebetween inclusive. [00156] In non-limiting embodiments, the method includes administering to a patient an antigen binding molecule, such as a humanized antigen binding molecule, as described herein having an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive. In non-limiting embodiments, the sequence may be varied, so long as the CD4 binding site remains intact and/or able to bind CD4. [00157] In non-limiting embodiments, the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. In non-limiting embodiments, the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. In non-limiting embodiments, the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. [00158] In non-limiting embodiments the antigen binding molecule useful in a method described herein includes a paratope having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), or a sequence having at least 70%, 75%. 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to any of the foregoing, all values and subranges therebetween inclusive. 33 66H6137.DOCX Attorney Docket No.06527-2502271 [00159] In view of the methods (of providing prophylaxis and/or treatment) described herein, which may include administering an antigen binding molecule (such as a humanized antigen binding molecule) described herein to a patient, also provided herein is a composition including an antigen binding molecule (such as a humanized J3 derivative antigen binding molecule) in a pharmaceutically-acceptable excipient, vehicle, or carrier. In non-limiting embodiments, the antigen binding molecule may have an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive. In non-limiting embodiments, the composition may be formulated for parental administration to a patient. [00160] Also provided herein are chimeric antigen receptor (CAR) T cells expressing an antigen binding molecule as described herein. [00161] A CAR may include a targeting moiety (e.g., an antigen binding molecule) for binding a surface antigen of another cell, and a signaling moiety for generating a signal when the targeting moiety binds a surface antigen on another cell. A CAR-T cell as described herein may include one or more CARs directed to one or more different surface antigens (target antigens) of a target, for example and without limitation, a B cell, that only activates the CAR-T-cell on which the binding molecules are expressed if the CAR(s) are bound to their target antigen. The CARs may be expressed on the surface of an engineered T cell, e.g., a CAR-T cell, therefore providing a CAR-T receptor that recognizes one or more surface antigens on a B-cell and is only activated when both antigens are present on a target (ABC) cell. A first target antigen may be B-cell maturation antigen (BCMA), CD33, CD19, and/or the like known to those of skill in the art. Methods of generating CAR-T cells are known to those of skill in the art, and may include generating a CAR construct that may include an antigen binding molecule, hinge and/or transmembrane domain, a costimulatory domain, and/or a signaling domain (and/or nucleic acids encoding any of the foregoing), delivering the construct to a T cell (autologous to a patient or otherwise) through transduction (e.g., lentiviral and/or retroviral transduction, electro- or nanoporation, or CRISPR/Cas9 systems), and expanding the cells. 34 66H6137.DOCX Attorney Docket No.06527-2502271 [00162] Using CAR-T cells to kill B cells or a subset of B cells and/or reducing the population of B cells or a subset of B cells, such as memory B cells, activated B cells, or ABC cells, may be useful in treating a variety of conditions. Relevant conditions include, but are not limited to, HIV, autoimmune diseases, and/or cancers (including, without limitation, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and/or multiple myeloma). As such, CAR-T receptors, constructed as earlier described may be designed to target surface antigens from B cells or a subset of B cells. For example, and without limitation, the surface antigen(s) may be for a memory B cell, the surface antigens may be for an activated B cell, or the surface antigens may be for an ABC cell. [00163] CAR-T cells expressing an antigen binding molecule as described herein may also be constructed or produced. CAR-T cells may be constructed or produced, in nonlimiting examples, by introducing genes for expressing antigen binding molecules into T cells. These T cells may be autologous or allogenic or xenogeneic. In non-limiting embodiments, the T cells are autologous or allogenic to a patient. In a non-limiting example, T cells may be collected and isolated from blood. In non-limiting examples, isolated T cells may be expanded ex vivo to increase the total number of T cells, such as by those methods described in Watanabe et al. (Watanabe N, et al. Impact of Manufacturing Procedures on CAR-T Cell Functionality. Front Immunol. 2022 Apr 13;13:876339). Genes for expressing antigen binding molecules may be introduced into the T cells ex vivo. Genes for expressing antigen binding molecules may be introduced into the T cells by a number of methods. For example and without limitation, T cells may be transfected or transduced with a nucleic acid or nucleic acids comprising a gene or genes for expressing antigen binding molecules. Transfection may be achieved by, for example and without limitation, physical means such as by electroporation, sonoporation, or other methods as are known in the art. Transfection may be achieved by, for example, by chemical means such as by using cationic lipids, cationic polymers, calcium phosphate, DAED-dextran, peptides, nanoparticles, or other methods as are known in the art. Transfection may be of a nucleic acid or nucleic acids comprising a gene or genes for expressing antigen binding molecules, for example as a plasmid or plasmids or with a gene editing system such as a CRISPR Cas9 system. Transduction may be achieved by, for example, lentiviral transduction, wherein a lentivirus inserts a nucleic acid or nucleic acids comprising a gene or genes for expressing CAR-T receptors into the genome of the host cell, such as a T cell. 35 66H6137.DOCX Attorney Docket No.06527-2502271 Other recombinant viral-mediated transfection vectors, such as AAV, HSV, or Ad vectors, as are broadly-known and available, may be employed for delivery of a nucleic acid comprising a gene for expression in a cell according to the methods described herein. Once constructed or produced, CAR-T cells may be expanded ex vivo to quantities sufficient for delivery into a patient in a dose effective, for example and without limitation, treat an autoimmune disease or reduce the population of B cells or subsets of B cells. [00164] For example and without limitation, a CAR-T cell may express one or more antigen binding molecules as previously described. CAR-T cells expressing one or more antigen binding molecules may, for example, be able to kill, in vitro or in vivo, a B cell or a subset of B cells, such as a memory B cells, activated B cells, or ABC cells. CAR-T cells may comprise a greater composition. A composition including CAR-T cells may include a pharmaceutically-acceptable excipient or carrier, such as water, PBS, or serum-free cell culture media. [00165] A composition may include one or more nucleic acids comprising a gene or genes for expressing one or more antigen binding molecules. In non-limiting examples, a composition may include a plasmid or plasmids comprising a gene or genes for expressing one or more antigen binding molecules. A nucleic acid or nucleic acids, such as a plasmid or plasmids, may comprise a gene or genes for expressing one or more antigen binding molecules comprising, for example a targeting moiety for binding a surface antigen of another cell, and a signaling moiety for generating a signal when the targeting moiety binds a surface antigen on another cell. Non-limiting examples of targeting moieties and signaling moieties for antigen binding molecules are previously described in this application, and any nucleic acid or nucleic acids comprising a gene or genes for expressing one or more antigen binding molecules may comprise, but are not limited to, any of the previously described moieties or those included in other examples found in this application. [00166] Any of the mentioned compositions may be useful in in vitro applications or in vivo applications. Examples of in vivo applications include, without limitation, reducing the population of B cells or subsets of B cells in a patient and/or treating patients. Such patients may include, but are not limited to, those with an autoimmune disease. For example and without limitation, one may treat an autoimmune disease in a patient with a previously described composition comprising CAR-T cells. CAR-T cells may be administered in one or more doses and may be administered in an amount 36 66H6137.DOCX Attorney Docket No.06527-2502271 effective to treat the autoimmune disease. Such amounts may range from, without limitation and inclusive of any increment therebetween, 102 to 1012 CAR-T cells. In another example, one may reduce the population of B cells or subsets of B cells in a patient with a previously described composition comprising CAR-T cells. CAR-T cells may be administered in one or more doses and may be administered in an amount effective to reduce the population of B cells or subsets of B cells in a patient. Such amounts may range from, without limitation and inclusive of any increment therebetween, 102 to 1012 CAR-T cells. In non-limiting examples, CAR-T cells may be delivered regionally or systemically such as through arterial infusion, intraventricular infusion, intratumoral infusion, intraperitoneal infusion, intrapleural infusion, or intravenous infusion. Other methods of delivery may include via a hydrogel or microneedle array. Non-limiting examples of methods of delivery may be found in Gu et al. (Gu, X., et al. Infusion and delivery strategies to maximize the efficacy of CAR-T cell immunotherapy for cancers. Exp Hematol Oncol 13, 70 (2024).). [00167] In non-limiting embodiments, a CAR-T cell as described herein may include a receptor (e.g., an antigen binding molecule) having an amino acid sequence having at least 70%, 75%. 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive. [00168] Also provided herein is A B cell engineered to express an antigen binding molecule as described herein. [00169] A B cell (e.g., a B lymphocyte) is a type of blood cell that acts as a center for a population of cells that express clonally diverse cell surface immunoglobulin (Ig) receptors recognizing specific antigenic epitopes (see, e.g., LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood.2008 Sep 1;112(5):1570-80). “B cells” include mature B cells, including subtypes thereof, such as, for example and without limitation, transitional B cells, naïve B cells, activated B cells, plasma B cells and memory B cells and, in the context of the present invention, precursors thereof, so long as such precursors comprise suitable surface antigens and/or other phenotypic or genotypic characteristics such that they may be subject to regulation as described herein. Activated B cells result from the binding of an antigen to a B cell 37 66H6137.DOCX Attorney Docket No.06527-2502271 receptor, such as the binding of an antigen to a B cell receptor of a naïve B cell. B cell activation may be done independent of T cells or through T cell dependent pathways. Memory B cells are a long-lived subset of B cells that, after initial activation, circulate throughout the body until they encounter an antigen that binds a B cell receptor on the memory B cell. After the B cell receptor is bound, a memory B cell may differentiate into a plasma cell. All genes and surface markers, as described herein, are broadly- known and do not require further explanation, such as B-cell markers, including, but not limited to CD19, 20, 21, 22, 23, 24, 25, 40, 72, CD79a,b, CD80, and/orCD86 (see, e.g. LeBien TW, et al. Blood.2008 Sep 1;112(5):1570-80). A “CD11c+ B cell” refers to a heterogenous population of B cells that express the CD11c surface antigen. They comprise mainly memory B cells, but may comprise other B cell types (see, e.g., Golinski ML, et al. CD11c+ B Cells Are Mainly Memory Cells, Precursors of Antibody Secreting Cells in Healthy Donors. Front Immunol. 2020 Feb 25;11:32). Age associated B cells (ABCs or ABC cells), may be expanded in the elderly, and also can be associated with various autoimmune diseases, and therefore provide therapeutic targets of the therapies and therapeutic compositions provided herein. ABC cells may be defined as a B cell subset characterized by the CD19+ (see, e.g., Wang K, et al. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol.2012 Nov 29;1(1):36), CD21− (see, e.g., Suryani S, et al. Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells. Blood. 2010 Jan 21;115(3):519-29), CD11c+ (see, e.g., Golinski ML, et al. Front Immunol.2020 Feb 25;11:32), T-bet+ (see, e.g., Knox JJ, et al. T-bet+ memory B cells: Generation, function, and fate. Immunol Rev. 2019 Mar;288(1):149-160) phenotype. [00170] B cells may be engineered to express an antigen binding molecule as described herein by introducing a construct (for example, including a promoter, coding sequence encoding the antigen binding molecule, a marker for selection, and/or a sequence for CRISPR-mediated knock-in) into the genome of the B cell (e.g., through a vector (such as a lentiviral vector, an AAV vector), electro- or nanoporation, mRNA transfection, and the like known to those of skill in the art) a nucleic acid encoding an antigen binding molecule. Cells engineered to express the antigen binding molecule can then be selected by known methods (e.g., fluorescence and/or antibiotic resistance). In non-limiting embodiments, the antigen binding molecule that is expressed has an amino acid sequence having at least 70%, 75%.80%, 85%, 90%, 38 66H6137.DOCX Attorney Docket No.06527-2502271 95%, 99%, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20 (including combinations thereof), all values and subranges therebetween inclusive. In non-limiting embodiments, the B-cell may be a B-cell that is autologous to a patient. In non-limiting embodiments, the engineered B-cell may then be reintroduced to the patient. Example Methods [00171] Antibody–antigen structural complex generation [00172] The COSMIC2 server (https://cosmic-cryoem.org/), a cloud-based computational platform developed by the University of Michigan for structural biology projects and openly accessible to academic researchers, facilitated the generation of an antibody–antigen structural complex. Initially, the protein sequence for gp120 Clade C was obtained from the Protein Data Bank (PDB: 7ri1) (https://www.rcsb.org/) and uploaded onto COSMIC2, along with the sequence files for HuJ3 and gp120 provided in the Supplementary Material. Subsequently, an AlphaFold2 job was initiated on the server. Since the AlphaFold toolkit on COSMIC2 does not provide configuration of the number of generated models, the “number of predictions per model” was set to 10 to enhance prediction diversity. [00173] Structure refinement and local docking of antibody–antigen complex [00174] The initial binding pose generated by AlphaFold-Multimer was refined using SnugDock ensemble docking. Due to the inaccuracy of AlphaFold-Multimer in modeling protein secondary structures, crystal structure of gp120 from PDB:7ri1 and the 3D structure of HuJ3, modeled by SWISS-MODEL (template PDB: 7ri1), were aligned to the complex generated by AlphaFold-Multimer and substituted for their corresponding components. This refined binding pose was then used as the input for SnugDock. The FastRelax function in Rosetta was employed to resolve potential clashes and identify energetically favorable conformations of the crystal gp120 and modeled HuJ3. Ten decoys were generated for both the crystal gp120 and modeled HuJ3, which were used as ensembles to enable flexibility of their backbone. These 10 relaxed gp120 and HuJ3 structures were prepacked using the Rosetta Prepack function to ensure low-energy starting side-chain conformations. Subsequently, the 39 66H6137.DOCX Attorney Docket No.06527-2502271 SnugDock function was used to dock the crystal gp120 ensemble with the modeled HuJ3 ensemble. SnugDock initiated the docking with a random perturbation of 3 Å translation and 8° rotation in each Cartesian direction. The Motif Dock Score was utilized during the low-resolution docking phase. One thousand decoys were generated and ranked by their interface score(I_sc). The formation of the docking funnel involved the criteria of whether the local docking was successful or not. Once the local docking was successful, the lowest I_sc result was chosen as the final local docking result. [00175] Computational alanine scanning and point mutation [00176] Possible hotspots (or key residues) on the antibody were predicted by Rosetta alanine scanning program (AlaScan.xml, https://github.com/Kortemme- Lab/ddg/). The distance cutoff value of interface residues was set to 5 Å. Among the alanine scanning results, the residues whose ΔΔG higher than 1 kcal/mol were selected as hotspots on the antibody. [00177] The “define_interface.py” was used to find out the interface residues on antibody. FlexddG program (https://github.com/Kortemme-Lab/flex_ddG_tutorial) was applied to perform single point mutation on the antibody interface residues to increase the binding affinity of HuJ3-gp120 complex. The complex we wished to mutate was placed in the “inputs” folder. The antibody chain id was specified in the “chains_to_move.txt” file located in the “inputs” folder. FlexddG only allows users to mutate one residue at a time, so the “run_example_2.py” script was modified to specify the residue chain id and number we wanted to mutate each time. The parameters used to run the program were set as the recommended values. The “analyze_flex_ddG.py” file was used to analyze the output results from “run_example_2.py”, which printed the wild type and mutant interface ΔG. ΔΔG score and the ΔΔG score reweighted with the fitted GAM model were calculated and listed in a .csv file through “analyze_flex_ddG.py”. The mutant whose reweighted ΔΔG score was lower than 0 kcal/mol was accepted as the mutation which may increase the binding affinity of the complex. [00178] BioLuminate point mutation validation [00179] BioLuminate software was downloaded from the Schrödinger website. The target complex structure was first imported to BioLuminate. Then, “protein preparation and refinement” was used to fix the structure (optimize orientations of hydrogen- bonded groups, delete waters, and minimize the structure). The parameters used the 40 66H6137.DOCX Attorney Docket No.06527-2502271 default setting. The prepared structure was then imported to the “Residue Scanning” panel and the “Calculation type” selected the “Stability and Affinity” mode. In the residues table, the residues predicted by our protocol were selected and mutated to the potential mutations. The other parameters used the default settings. The mutations with affinity scores smaller than 0 kcal/mol were accepted. [00180] Humanization of nanobody J3 [00181] Llama nanobody J3 was humanized by grafting its complementarity determining regions (CRDs) (CDR1–3) residues to the closest human Ig germline variable domain (VH) family VH3–23 scaffold. However, not all framework region (FR) residues are human. We deliberatively kept specific llama-derived FR residues, which include the vennerier region and packing residues that can modulate CDRs conformations and impact antigen binding. In addition, the “tetrad” in FR2, which is important to the nanobody stability and solubility, was left in its native state. Through this process, we obtained huJ3 with a 13 amino acid difference from the original nanobody J3. The “humanness” prediction results indicated the HuJ3 displayed a higher sequence similarity to naïve human antibody repertoire than the original J3, indicating a reduced risk of inducing immunogenicity in human applications. [00182] Experimental validation of point mutation [00183] All of the mutations of HuJ3 were selected for experimental validation. Their mutation sequence alignment was shown in supporting. These mutants were recombinantly expressed by the phagemid pComb3x, PComb3x was modified based on the original plasmid pComb3XSS, which is purchased from Addgene (Plasmid #63890). In the pComb3X, the lac promoter is used for transcription initiation followed by an OmpA signal peptide directing VH proteins secretion into periplasm. The six consecutive histidine tag and the following Flag tag were used for protein purification and detection. The pComb3x containing huJ3 was constructed by molecular subcloning to insert the huJ3 gene into the Sfi I linearized pComb3x backbone using the enzymes Sfi I and T4 ligase. The mutants plasmids were made through site- directed mutagenesis at BonOpus. Competent HB2151 Escherichia coli was transformed with these plasmids to express VH nanobodies. Antibodies were purified from E. coli periplasm after polumyxin B treatment followed by Ni-NTA chromatography. The molecular size and purity was verified by SDS-PAGE. ELISA was performed to determine quantitative binding of each variant to the HIV-1 envelope glycoprotein. Transiently expressed gp140, a functional homolog of gp120 used to 41 66H6137.DOCX Attorney Docket No.06527-2502271 evaluate HIV-1 binding in vitro, served as the antigen for evaluation. Plates were coated with 50 ng gp140/well at 4°C overnight and subsequently blocked with 3% milk for 1 h at room temperature. Primary VH nanobodies, including the original J3, HuJ3, and each of the five variants, were serially diluted in 3% milk to achieve an 8- concentration gradient ranging from 1000 to 0.0128 nM, then incubated on the coated and blocked plate for 2 h at room temperature. After incubation, the plate was washed 4 times by 0.05% PBST. The detection antibody, anti-FLAG M2-peroxidase (A8592, Sig-ma-Aldrich, St. Louis, MO, USA), was added and incubated for 1 h at room temperature. The plate was washed another 4 times by 0.05% PBST. Binding activity was detected using 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped after 2 min by TMB stop buffer (ScyTek Laboratories, Logan, UT, USA) to prevent oversaturation and absorbance was read at 450 nm. The experiment was performed in duplicate. After analysis, another ELISA was performed in the same manner as above to validate and compare the gp140 affinity of variant E44R with J3 and HuJ3. [00184] E44R was further validated in the laboratory-developed TZM-bl HIV-1 Phenotyping Assay to measure the susceptibility of HIV-1 (group M, subtype B, isolate BAL) to E44R compared with HuJ3. TZM-bl cells are an indicator cell line that allows quantitative analysis of HIV replication. TZM-bl cells were generated from HeLa cells that stably express large amounts of CD4 and CCR5 and have separately integrated copies of the luciferase and ß-galactosidase genes under control of the HIV-1 promoter. These cells naturally express CXCR4 receptors. TZM-bl cells were plated at 10,000 cells per well overnight. The cells were treated with serial dilutions of HuJ3 antibodies, and infected with a dilution of infectious HIV-1 virus normalized to an output of 140,000 relative light units (RLU) as determined by endpoint dilution. After a 48-h incubation at 37°C, cells were lysed, and luminescence was measured in RLU using a commercially available luciferase detection system. The 50% in vitro concentration (IC50) was calculated as the concentration of antibodies needed to inhibit 50% of HIV- 1 replication in the assay. A batch control virus was run with each experimental setup. [00185] HuJ3 designed by IsAb1.0 [00186] The crystal structure of gp120 CladeC (PDB: 7ri1) was obtained from the PDB (https://www.rcsb.org/). The sequence of HuJ3 was submitted to the SWISS- MODEL web server (https://swissmodel.expasy.org/interactive). VHH nanobody J3 (PDB: 7ri1) was selected as the template to model the HuJ3. 42 66H6137.DOCX Attorney Docket No.06527-2502271 [00187] ClusPro was employed to generate the potential binding poses of the HuJ3- gp120 complex. To specify the antibody docking, “Antibody Mode” was chosen. HuJ3 was designated as the receptor and gp120 as the ligand. Paratopes and epitopes were entered into the attraction sections of the receptor and ligand, respectively, to set the docking constraints. The cluster among the top 10 global docking results that closely resembled the binding pose of PDB: 7ri1 was selected as the potential binding pose. [00188] The selected binding pose from global docking was input into the SnugDock function on the ROSIE web server, utilizing “thorough mode” to refine the structure. The success of the local docking is based on the formation of the docking funnel. Once the local docking was successful, the lowest I_sc result was chosen as the final local docking result. [00189] Potential hotspots (or key residues) on the antibody were identified using the Rosetta alanine scanning program (AlaScan.xml). The distance cutoff for interface residues was set to 5 Å. Among the alanine scanning results, residues with a ΔΔG higher than 1 kcal/mol were identified as hotspots on the antibody. [00190] Single State Design protocol was obtained from Dr. Jens Meiler’s lab website. Initially, ‘define_interface.py’ was utilized to prepare a residue file (resfile), which defined the interface residues should be mutated. Subsequently, the Single State Design protocol (design.xml) was employed to modify the antibody. Results [00191] fdf Workflow of IsAb 2.0 [00192] The general procedure of IsAb2.0 is outlined in FIG. 1. In the first step, users input sequences of the antibody and antigen into the protocol, with antibody sequences retrievable from the IMGT (https://www.imgt.org/) database. In Step 2, AlphaFold-Multimer2.3/3.0 generates 3D structures of the antibody, antigen, and their complex, effectively combining the functions of homology modeling and global docking from IsAb1.0. The per-residue confidence metric (pLDDT) is used to evaluate the quality of the model generated by AlphaFold-Multimer, assessing local accuracy. If the pLDDT scores are below 70, the antibody–antigen complex undergoes further refinement. Otherwise, the complex proceeds to Step 4 for local docking. In Step 3, several methods can be used for structural refinement. If the structure with lower pLDDT score has a crystal structure, this crystal structure will be optimized by Rosetta FastRelax to resolve potential clashes and identify energetically favorable conformations. The relaxed structures will then replace the model generated by 43 66H6137.DOCX Attorney Docket No.06527-2502271 AlphaFold-Multimer. If a crystal structure is unavailable, SWISS-MODEL will generate a new 3D homology model for low pLDDT structure and replace it. In Step 4, SnugDock performs local docking for the antibody and antigen, allowing flexibility of the CDR loops and interfacial side chains. SnugDock refines the potential binding poses provided by AlphaFold-Multimer and outputs the final antibody–antigen complex. In Step 5, after obtaining the 3D structure of antibody–antigen complex, alanine scanning is performed to predict possible hotspots on the antibody, facilitating future antibody design. Alanine scanning mutates the interface residues to alanine and calculates the change in energy to identify hotspots. In Step 6, point mutations are applied to mutate the antibody interface residues to the remaining 17 amino acids and identify mutations that can improve the binding affinity of the complex. The antibody– antigen complex is imported into FlexddG to perform point mutations, identifying mutations that enhance binding affinity. [00193] Antibody–antigen structural complex modeling [00194] As a result, we acquired 50 decoys along with their respective predicted local distance difference test (pLDDT) scores for subsequent analysis. By ranking the decoys based on their interface predicted template modeling and predicted template modeling (ipTM+pTM) scores, we identified the top 10 models with the highest scores. Remarkably, nine of these top models shared an identical binding position with J3- gp120. The model with the highest pLDDT and ipTM + pTM scores was selected as the likely binding pose. However, upon aligning the gp120 modeled by AlphaFold- Multimer with the crystal gp120, it was revealed that even the best model, Model_3_Pred_5, contained some structural errors. These errors included a loss of alpha helix between residues LEU369-THR372 and two structural errors in the beta- sheet regions, specifically between residues LYS419-LYS421 and ASP464-GLU466. The pLDTT plot indicated that all these regions had lower pLDTT scores. For HuJ3, since no crystal structure is available, the model generated by AlphaFold-Multimer was evaluated based on the pLDDT scores. According to the pLDTT plot, the pLDTT value in CDR1 between residues PHE29-GLN31 was lower than 60, indicating low confidence in this region. Structure analysis suggested that AlphaFold-Multimer may have failed to predict the alpha helix in this region. Similarly, the CDR3 residues SER105-GLY107 also had lower pLDTT scores. The structural analysis showed that this region may have failed in modeling an alpha helix. Since both CDR1 and CDR3 are important in the local docking step. Hence, structure refinement was conducted to 44 66H6137.DOCX Attorney Docket No.06527-2502271 correct the structure of these regions. Additionally, Table 1 (below) summarizes the average pLDDT and ipTM+pTM scores for each model. Moreover, detailed pLDDT data for each residue was plotted, enhancing the comprehensive overview of model performance metrics: Table 1 [00195] HuJ3-gp120 local docking [00196] AlphaFold-Multimer encountered challenges in accurately modeling protein secondary structures. Consequently, the crystal structures of gp120 and modeled HuJ3 generated by SWISS-MODEL were used to replace the corresponding in the complex modeled by AlphaFold-Multimer. Then, using the complex binding pose to serve as starting structure to the local docking. The SnugDock ensemble docking was employed to perform local docking searches using backbone ensembles, which has been proved to improve docking accuracy. This ensemble docking approach relies on the conformational-selection mechanism for protein docking, utilizing a pre-generated ensemble of protein partners. The ensembles were generated using Rosetta Relax to sample various backbone conformations during docking. In the low-resolution stage, each docking run involves rigid-body translation and rotation around the protein partner, along with backbone swapping from the pre-generated ensemble. This allows for the sampling of diverse backbone conformations. In the high-resolution stage, an all-atom refinement is applied to the generated encounter complex, and the side- 45 66H6137.DOCX Attorney Docket No.06527-2502271 chains at the interface are packed for optimal binding. The interface root means square deviation (I_rmsd) of heavy atoms in the interface residues between the reference structure and resulting structure was used to evaluate the performance of local docking. According to the Critical Assessment of Protein Interactions, docking results with an I_rmsd value lower than 4 Å are considered “near-native” structure. According to the criteria from SnugDock, the robust evaluation of successful local docking is the presence of a “docking funnel”, in which the “near-native” result (low I_rmsd) has lower energy (low I_sc) than the non-native result. In this case, three of the five lowest interface score (intermolecular energy/I_sc) results have an I_rmsd value lower than 4 Å (N5 > =3), and the performance will be identified as successful docking. From the I_sc versus I_rmsd plot, we found that all the five lowest I_sc results had I_rmsd lower than 4 Å, which meant that this local docking was successful and the native HuJ3- gp120 binding pose has a higher chance of being similar to the binding pose we predicted. As for the final result, we chose the lowest I_sc result as our final binding pose of the HuJ3-gp120 complex. The predicted HuJ3-gp120 complex also overlapped with the J3-gp120 crystal structure and showed that the binding pose of HuJ3 to gp120 was highly similar to J3 (FIG.2). [00197] After the structural analysis of CD4-gp120 and J3-gp120, we found that residues D368, N425, M426, and V430 on gp120 interacted with both J3 and CD4. In this case, we proposed that these residues may be the epitopes on gp120. In addition, the binding site on gp120 of J3 and HuJ3 are the same as CD4. Therefore, we hypothesized that these epitopes on gp120 will form a connection with HuJ3. Structural analysis of the predicted HuJ3-gp120 complex (FIG.3) revealed that gp120 formed 12 hydrogen bonds with HuJ3 (Table 2, below): 46 66H6137.DOCX Attorney Docket No.06527-2502271 Table 2 [00198] The structure analysis of the HuJ3-gp120 complex revealed that D368 on gp120 formed hydrogen bonds with H56 and I101 (HuJ3). N425, M426, and V430 formed hydrogen bonds with Y100, T99, and N30 on HuJ3, respectively. Compared with FIG.3, panels B and D, we noticed that both these interactions could be found on the J3-gp120 and CD4-gp120 complexes, which matched with the hypothesis we made previously and increased the reliability of the HuJ3-gp120 complex we predicted. [00199] HuJ3-gp120 alanine scanning and point mutation 47 66H6137.DOCX Attorney Docket No.06527-2502271 [00200] Computational alanine scanning was applied to predict the hotspots on the HuJ3-gp120 interface. Based on the criteria that neutral residues and hotspots were defined as ΔΔG less or more than 1 kcal/mol during the mutation to alanine, six residues were defined as possible hotspots (Table 3, below): Table 3 [00201] To perform point mutation for the HuJ3-gp120 complex, the ‘define_interface.py’ was used to find the interface residues on HuJ3. The interface cutoff value was defined as 5 Å, and 18 residues on HuJ3 were identified as interface residues. Since previous experiments have tested mutations on CDR1 and Framework Region 1, our current focus is on mutating CDR2, CDR3 and interface residues. All these residues were input into FlexddG program and mutated to the remaining 17 amino acids. The mutants whose ΔΔG smaller than 0 kcal/mol were filtered from the FlexddG results, and E44, V54, S98, T99, and S102 were all identified as possible mutation sites. Structure rational analysis was used to reduce the number of possible mutations. ΔΔG of E44R was −0.394 kcal/mol, the second lowest mutation among the other E44 substitutions. Given that E44 is located within a negatively charged pocket, mutating it to a positively charged residue could enhance electrostatic interactions favorably (FIG.4A, panel A). V54E had ΔΔG value −0.299 kcal/mol and E54 formed two hydrogen bonds with R432 on gp120 (FIG.4A, panel B). Because S98 was in a hydrophobic pocket, it mutated into a hydrophobic residue which could imbue the complex with better electrostatic properties (FIG.4B, panel C). T99R had the lowest ΔΔG value −0.979 kcal/mol among the other substitutions. Compared with T99 forming a hydrogen bond with gp120, R99 formed two hydrogen bonds with W427 and one with D474, which made the binding between HuJ3 and gp120 more stable (FIG. 48 66H6137.DOCX Attorney Docket No.06527-2502271 4B, panel D). S102R connected with P470 and I371 on gp120 by forming a hydrogen bond with them respectively, its ΔΔG value −1.752 kcal/mol was the lowest among the other substitutions (FIG. 4C). Overall, E44R, V54E, S98W, T99R, and S102R were selected as the potential mutations. [00202] To increase the reliability of our predictions, the powerful protein engineering software BioLuminate from Schrödinger was applied to test the five predicted mutants. Each potential mutation was performed by BioLuminate again. According to BioLuminate result analysis rules, the ‘Δ Affinity’ score lower than zero means the mutant binds more effectively than the original protein. Four of the five mutations predicted by our protocol with ‘Δ Affinity’ lower than 0 (Table 4, below), indicating that four of the five mutations predicted by the protocol and BioLuminate had the same results. Table 4 [00203] ELISA validation (FIG. 5, middle panel) showed that E44R had better binding affinity than HuJ3, which is consistent to the predicted result. V54E had the same binding affinity as HuJ3. The T99R mutant protein failed to express. S98W and S102R exhibit lower binding affinity than HuJ3. HIV-1 neutralization was consistent with ELISA results, with E44R showing an enhanced neutralization potency compared with HuJ3. However, its neutralization capacity was still lower than that of the original J3 nanobody (Table 5, below): 49 66H6137.DOCX Attorney Docket No.06527-2502271 Table 5 [00204] A potential reason why V54E, S98W, and S102R failed to increase the binding affinity is that the side chain of valine and serine are uncharged, but glutamic acid and arginine have charged side chains. Also, tryptophan is significantly larger than serine. Such mutations may have caused a repulsive effect. Additionally, the loop regions are flexible, which may change the conformation of the loops, leading to decrease or no change to the binding affinity when mutations are introduced. As for T99R, it is likely that the mutation caused inaccurate folding and led to expression failure. [00205] Comparisons between IsAb2.0 and IsAb1.0 [00206] To demonstrate the improvement of IsAb2.0, IsAb1.0 was applied to design HuJ3 and its predictions validated by the experimental methods. FIG.6 illustrates that the differences between the 3D structures of HuJ3-gp120 complexes generated by IsAb1.0 and IsAb2.0 were minimal. The modeling and docking results from both versions were closely aligned. However, IsAb2.0 does not require modeled structures to have homologous proteins as template, allowing it to generate 3D structures for novel antibodies and antigens. Additionally, IsAb2.0 does not require pre-existing binding information of the antibody and antigen, making it broadly applicable in various scenarios. The integration of AlphaFold-Multimer in IsAb2.0 enables the direct generation of antibody–antigen complexes from their sequences, achieving similar accuracy to complexes generated by IsAb1.0. This use of AlphaFold-Multimer effectively replaces the processes of homology modeling and global docking in IsAb1.0, significantly simplifying the protocol. FIG. 5, top panel shows that all four mutations predicted by IsAb1.0 exhibited either the same or lower binding affinity compared with HuJ3. In contrast, as discussed previously, one of the five predictions 50 66H6137.DOCX Attorney Docket No.06527-2502271 from IsAb2.0 was shown to increase the HuJ3-gp120 binding affinity, indicating that IsAb2.0 achieves higher prediction accuracy than IsAb1.0. Since protein–protein interaction can cause structure changes, Single State Design does not account for the conformational plasticity of proteins, leading to inaccurate predictions. Hence, FlexddG that uses the backrub method to generate ensemble models and allows structure flexibility can achieve higher accuracy in increasing antibody binding affinity. Discussion [00207] In this study, we developed an advanced antibody design protocol, IsAb2.0, by integrating state-of-the-art AI-based and physical methods. IsAb2.0 can be utilized for the design of humanized antibodies and nanobodies, enabling the construction of more accurate models of antibody–antigen complexes without the need for template and additional binding information, using AlphaFold-Multimer. Furthermore, IsAb2.0 employs more accurate method FlexddG for predicting mutations. To validate our protocol and apply J3 to treat HIV-1 in human patients, we first humanized the llama nanobody J3 to HuJ3 to decrease its immunogenicity. We then applied the IsAb2.0 to design HuJ3 and increased its binding affinity with gp120. The IsAb2.0 modeled HuJ3- gp120 complex. Based on the HuJ3-gp120 complex, the IsAb2.0 predicted six hotspots on HuJ3 and found five potential mutations that could increase the binding affinity of the HuJ3-gp120 complex. All five mutations had been repeated by BioLuminate, and four of the five mutations gave the same results as our protocol, showing that IsAb2.0 yielded similar predictions as the well-known commercial protein design software. Among the predicted mutations, experimental validation by ELISA proved that E44R could increase the binding affinity of HuJ3-gp120. HIV-1 neutralization assays also showed the increased neutralization capacity of E44 compared with HuJ3. Although the prediction accuracy did not reach our expectations, these results support that IsAb2.0 has the potential to be applied to antibody design, including the design of nanobodies and humanized antibodies. In the future, we will further refine our protocol and resolve its limitations. For example, we will aim to improve the accuracy of point mutation which, at this point, is not sufficient. This may be due in part to the inaccuracies of the score function in FlexddG. The current score function may not evaluate the mutations accurately, leading to point mutation failure. Also, the current point mutation program does not consider the rationale of mutations, which contributes to the failure of prediction. The process of running FlexddG is complicated, leading to a prohibitively expensive computing time. Another limitation of 51 66H6137.DOCX Attorney Docket No.06527-2502271 our protocol is that it cannot run automatically. In some steps, to maintain prediction accuracy, it still requires users to choose the results manually. This problem means the protocol is not yet entirely user-friendly, especially for users who are not experienced in the area of antibody engineering. [00208] To solve the limitations mentioned above, we will develop a novel score function suitable for FlexddG that enhances the precision of the result evaluation. We will also examine the rationale of amino acids substation during mutation. In addition, we will create a machine-learning-based method than can predict amino acids probability in each position based on the difference between the framework and CDR regions. This method will combine with FlexddG and build an advanced program. The advanced program will let FlexddG mutate the specified position to the amino acids with high occurrence probability which can improve the efficiency of FlexddG. Another solution is to leverage the advantages of graph neural networks in handling 3D structures to develop an AI-based antibody design model. This AI-based model can more effectively represent the interactions between antibodies and antigens, enabling it to learn the patterns of the interactions within complexes and design the antibodies with higher accuracy. To achieve the automation of the protocol and maintain prediction accuracy, we will create more advanced programs or modify existing methods to let the protocol select the correct results by itself. [00209] Another limitation is that AlphaFold-Multimer often fails to accurately model secondary structures, which can affect local docking accuracy. Users can evaluate the quality of structures modeled by AlphaFold-Multimer based on the pLDDT scores of residues. According to the AlphaFold official document, regions with pLDDT scores lower than 70 have low confidence and should be treated cautiously. We suggest that if pLDDT scores of all regions, or the interaction critical regions, are higher than 70, users can directly input complex modeled by AlphaFold-Multer into SnugDock. In our case, some secondary structures of both the antibody and antigen were not accurately predicted. Hence, structure refinement is necessary to achieve higher structural accuracy for the antibody or antigen. The best way to perform structure refinement is to replace the structures of both the antibody and antigen modeled by AlphaFold- Multimer with high-resolution crystal structures. If the crystal structures are unavailable, users can generate accurate 3D structures by some powerful modeling program, e.g. SWISS-MODEL. 52 66H6137.DOCX Attorney Docket No.06527-2502271 [00210] Overall, the improvements made to the IsAb2.0 protocol has important implications for the future of antibody design. A standard, user-friendly antibody design tool could significantly improve and accelerate the development of antibody therapies across several fields, including the treatment of cancers, viral infections, and addiction. By developing IsAb2.0 protocol to include methods of nanobody and humanized antibody design while reducing its reliance on available structural data, we aim to expand treatment options for such conditions and reduce barriers to antibody engineering. [00211] Having described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. References incorporated herein by reference are incorporated for their technical disclosure and only to the extent that they are consistent with the present disclosure. 53 66H6137.DOCX

Claims

Attorney Docket No.06527-2502271 THE INVENTION CLAIMED IS 1. A humanized J3 derivative antigen binding molecule. 2. The humanized antigen binding molecule of claim 1, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. 3. The antigen binding molecule of claim 2, wherein the amino acid sequence comprises SEQ ID NO: 14. 4. The antigen binding molecule of claim 2, wherein the amino acid sequence consists of SEQ ID NO: 14. 5. The humanized antigen binding molecule of claim 1, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. 6. The antigen binding molecule of claim 5, wherein the amino acid sequence comprises SEQ ID NO: 3. 7. The antigen binding molecule of claim 5, wherein the amino acid sequence consists of SEQ ID NO: 3. 8. The humanized antigen binding molecule of claim 1, having an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. 9. The antigen binding molecule of claim 8, wherein the amino acid sequence comprises SEQ ID NO: 9. 10. The antigen binding molecule of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 9. 54 66H6137.DOCX Attorney Docket No.06527-2502271 11. A humanized antigen binding molecule comprising a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. 12. A method of binding or neutralizing a human immune deficiency virus (HIV) particle, comprising contacting the HIV particle with a humanized J3 derivative antigen binding molecule. 13. The method of claim 12, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. 14. The method of claim 13, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. 15. The method of claim 13, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. 16. The method of claim 12, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. 17. The method of claim 16, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. 18. The method of claim 16, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. 19. The method of claim 12, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. 55 66H6137.DOCX Attorney Docket No.06527-2502271 20. The method of claim 19, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. 21. The method of claim 19, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. 22. The method of claim 12, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. 23. The method of claim 12, wherein the HIV particle is an HIV1 particle. 24. A method of treating a patient infected with HIV comprising administering to the patient a humanized J3 derivative antigen binding molecule to the patient in an amount effective to treat the infection. 25. The method of claim 24, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. 26. The method of claim 25, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. 27. The method of claim 25, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. 28. The method of claim 24, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. 56 66H6137.DOCX Attorney Docket No.06527-2502271 29. The method of claim 28, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. 30. The method of claim 28, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. 31. The method of claim 24, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. 32. The method of claim 31, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. 33. The method of claim 31, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. 34. The method of claim 24, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. 35. The method of claim 24, wherein the HIV is HIV1. 36. A composition comprising a humanized J3 derivative antigen binding molecule in a pharmaceutically-acceptable excipient, vehicle, or carrier. 37. The composition of claim 36, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 14. 38. The composition of claim 37, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 14. 57 66H6137.DOCX Attorney Docket No.06527-2502271 39. The composition of claim 37, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 14. 40. The composition of claim 36, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 3. 41. The composition of claim 40, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 3. 42. The composition of claim 40, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 3. 43. The composition of claim 36, wherein the antigen binding molecule has an amino acid sequence having at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, optionally at least 95%, optionally at least 99%, sequence identity to SEQ ID NO: 9. 44. The composition of claim 43, wherein the antigen binding molecule has an amino acid sequence comprising SEQ ID NO: 9. 45. The composition of claim 43, wherein the antigen binding molecule has an amino acid sequence consisting of SEQ ID NO: 9. 46. The composition of claim 36, wherein the antigen binding molecule comprises a paratope comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20. 47. The composition of claim 36, formulated for parental administration. 48. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 14. 58 66H6137.DOCX Attorney Docket No.06527-2502271 49. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 3. 50. A CAR-T cell expressing an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 9. 51. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 14. 52. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 3. 53. A B-cell engineered to express an antigen binding molecule comprising the amino acid sequence of SEQ ID NO: 9. 59 66H6137.DOCX
PCT/IB2025/058241 2024-08-13 2025-08-13 Humanization and affinity maturation of the cd4-mimicking llama nanobody j3 Pending WO2026038164A1 (en)

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