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WO2025054320A1 - Anticorps anti-ackr4, compositions et utilisations associées - Google Patents

Anticorps anti-ackr4, compositions et utilisations associées Download PDF

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Publication number
WO2025054320A1
WO2025054320A1 PCT/US2024/045377 US2024045377W WO2025054320A1 WO 2025054320 A1 WO2025054320 A1 WO 2025054320A1 US 2024045377 W US2024045377 W US 2024045377W WO 2025054320 A1 WO2025054320 A1 WO 2025054320A1
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Prior art keywords
sequence
seq
cdr
nos
antibody
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Inventor
Courtney BEERS
John Corbin
Vanessa Soros
Pamela ZHANG
Rashmi BANKOTI
Nicole MALANDRO
Kiran Ahluwalia
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Tizona Therapeutics
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Tizona Therapeutics
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • ACKR4 is a cell surface receptor with seven transmembrane domains structurally similar to G protein-coupled receptor proteins.
  • ACKR4 is a receptor for C-C type chemokines and has been shown to bind to dendritic cell and T cell-activated chemokines including CCL19, CCL21, and CCL25. [0005]
  • ACKR4 has been found to regulate the availability of chemokines. Specifically, unlike other G-protein coupled receptors, ACKR4 functions as a “scavenger,” internalizizing chemokines and sorting them for lysosomal degradation to limit local and systemic chemokine concentrations.
  • ACKR4 is an attractive target for regulating chemokine concentration and is also attractive as a therapeutic target.
  • a first aspect provides an antibody that binds specifically to a human ACKR4. 1107368.00126 The antibody is capable of one or more of inhibition of chemokine scavenging; ACKR4 internalization; and ACKR4 down-regulation.
  • the antibody is a monoclonal antibody.
  • the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody is a bispecific antibody, a multi-specific antibody, a diabody, or a multivalent antibody.
  • the antibody is of the IgG1, IgG2, IgG3, IgG4, IgA, or IgM type. In some embodiments, the antibody is an antigen-binding antibody fragment. In some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an Fv fragment. In some embodiments, the antibody is a single chain antibody, a single domain antibody, or a nanobody.
  • the isolated antibody comprises, consists of, or consists essentially of a heavy chain variable region (VH) and a light chain variable region (VL), the VH and/or VL comprising 1, 2, 3, 4, 5, or 6 of: a VHCDR1 having the sequence set forth in SEQ ID NOS: 1-5 or SEQ ID NOS: 12-16; a VHCDR2 having the sequence set forth in SEQ ID NOS: 23-25 or SEQ ID NOS: 34-36; and a VHCDR3 having the sequence set forth in SEQ ID NOS: 45-48; a VLCDR1 having the sequence set forth in SEQ ID NOS: 55-59; a VLCDR2 having the sequence set forth in SEQ ID NOS: 67-70, and a VLCDR3 having the sequence set forth in SEQ ID NOS: 77-81.
  • VHCDR1 having the sequence set forth in SEQ ID NOS: 1-5 or SEQ ID NOS: 12-16
  • VHCDR2 having the sequence set forth in SEQ ID NOS
  • the human ACKR4 comprises, consists of, or consists essentially of a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising: a VHCDR1 having the sequence set forth in SEQ ID NOS: 1-5 or SEQ ID NOS: 12-16, a VHCDR2 having the sequence set forth in SEQ ID NOS: 23-25 or SEQ ID NOS: 34-36, a VHCDR3 having the sequence set forth in SEQ ID NOS: 45-48; and the VL comprising: a VLCDR1 having the sequence set forth in SEQ ID NOS: 55-59, a VLCDR2 having the sequence set forth in SEQ ID NOS: 67-70, and a VLCDR3 having the sequence set forth in SEQ ID NOS: 77-81.
  • the isolated antibody molecule capable of binding to human ACKR4 comprises, consists of, or consists essentially of a heavy chain variable region (VH) and/or a light chain variable region (VL), the VH comprising at least one sequence set forth in any of SEQ ID NOS: 89-94 and the VL comprising at least one sequence set forth in any of SEQ ID NOS: 101-106.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody molecule capable of binding to human 1107368.00126 ACKR4 comprises, consists of, or consists essentially of a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising 1, 2, or 3 of: a VHCDR1 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 1-5 or 12-16, a VHCDR2 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 23-25 or SEQ ID NOS: 34-36, and a VHCDR3 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 45-48; and the VL comprising 1, 2, or 3 of: a VLCDR1 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 55-59, a VLCDR2 having an amino acid sequence that is at least 90% identical to the sequence set forth in
  • the antibody molecule capable of binding to human ACKR4 comprises, consists of, or consists essentially of a heavy chain variable region (VH) and a light chain variable region (VL), VH comprising 1, 2, or 3 of: a VHCDR1 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 1-5 or 12-16, a VHCDR2 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 23-25 or SEQ ID NOS: 34-36, and a VHCDR3 having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NOS: 45-48; and the VL comprising 1, 2, or 3 of: a VLCDR1 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 55-59, a VLCDR2 having an amino acid sequence that is homologous to the sequence set forth in SEQ ID NOS: 67-
  • antibody molecule capable of binding to human ACKR4 comprises, consists of, or consists essentially of a heavy chain and a light chain, the heavy chain comprising one or more molecules having a sequence consisting of one of SEQ ID NOS: 113-120 and the light chain comprising one or more molecules having a sequence consisting of one of SEQ ID NOS: 128-133.
  • the antibody molecule capable of binding to human ACKR4 comprises, consists of, or consists essentially of a heavy chain and a light chain, the heavy chain comprising one or more molecules, the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 113 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 128; the heavy chain comprising one or more molecules, each molecule having a sequence 1107368.00126 consisting of SEQ ID NO: 114 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 129; the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO: 115 and the light chain comprising one or more, each molecule having a sequence consisting of SEQ ID NO: 130; the heavy chain comprising one or more molecules, each molecule having a sequence consisting of SEQ ID NO:
  • a second aspect provides an isolated nucleic acid encoding an antibody provided herein.
  • the isolated nucleic acid comprises an expression vector.
  • Some embodiments provide a prokaryotic or eukaryotic host cell transformed with the one or more expression vectors.
  • Some embodiments provide an oncolytic virus encoding the nucleic acid.
  • Some embodiments provide a method for the production of an antibody of the invention comprising the steps of expressing a nucleic acid provided herein in a prokaryotic or eukaryotic host cell and recovering the protein from the cell or the cell culture supernatant.
  • a third aspect provides a pharmaceutical comprisition comprising any of the antibodies set forth herein. In some embodiments, the pharmaceutical composition is adopted to any suitable route of administration.
  • the pharmaceutical compistion comprises one or more excipients. Some embodiments further comprise parenteral dosage forms.
  • a fourth aspect provides a method of treating an individual having a disease or condition comprising administering any of the antibodies set forth herein to an individual in need thereof.
  • the disease or condition is cancer, autoimmune disease, and infection. 1107368.00126 BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG.1 provides a table showing recombinant ACKR4 micelle binding, cellular ACKR4 binding, and inhibition of chemokine scavenging by ACKR4 antibodies disclosed herein.
  • FIG. 2A shows CHO-hACKR4 binding by representative ACKR4 antibodies.
  • FIG.2B shows sensorgram of recombinant ACKR4 micelle binding by representative ACKR4 antibodies.
  • FIG. 3 shows CHO ACKR4 and ECL mutant cell binding by ACKR4 antibodies disclosed herein.
  • FIG. 4A and 4B show inhibition of CCL21 scavenging by representative ACKR4 antibodies in AsPC-1 WT and SUIT2 WT cells, respectively.
  • FIG. 4C shows inhibition of CCL19 scavenging by representative ACKR4 antibodies in SUIT2 WT cells.
  • FIG. 5A shows extracellular luminescence of HiBiT-ACKR4 upon treatment with ACKR4 antibody or control after 15 h.
  • FIG.5B shows % HiBiT-ACKR4 internalization by ACKR4 antibody calculated by: 100 X [1 - (RLU of treatment / RLU of Neg Ctl IgG)].
  • FIG.5C and 5D show time-dependent % HiBiT-ACKR4 internalization by ACKR4 antibodies disclosed herein.
  • FIG. 5E shows lytic luminescence of HiBiT-ACKR4 upon treatment with ACKR4 antibody or control after 15 h.
  • FIG.5F shows % HiBiT-ACKR4 down-regulation by ACKR4 antibody calculated by: 100 X [1 - (RLU of treatment / RLU of Neg Ctl IgG)].
  • FIG.6A shows in vivo detection of 20 ug of ACKR4 antibodies in mouse serum of BALB/c mice after seven days.
  • FIG.6B shows in vivo detection of 5 ug of ACKR4 antibody in mouse serum of huACKR4 KI/muACKR4 KO mice (HO) and muACKR4 wild type mice (WT) after seven days.
  • FIG. 6C shows in vivo detection of 250 ug of ACKR4 antibodies in mouse serum of HO mice after seven days.
  • FIG. 6A shows in vivo detection of 20 ug of ACKR4 antibodies in mouse serum of BALB/c mice after seven days.
  • FIG.6B shows in vivo detection of 5 ug of ACKR4 antibody in mouse serum of huACKR4 KI/muACKR4 KO mice (HO) and muACKR4 wild type mice (WT) after seven days.
  • FIG. 6C shows in vivo detection of 250 ug of ACKR4 antibodies in
  • 7A C57BL/6 mice
  • 7B HuACKR4 KI/muACKR4 KO mice
  • WT muACKR4 wild type mice
  • 7C HO mice
  • 7D C57BL/6 mice
  • 7E HO and WT mice
  • 7F HO mice
  • DETAILED DESCRIPTION 1107368.00126 Provided herein are antibodies that selectively bind to ACKR4 and compositions comprising the antibodies. Also provided are methods of using the antibodies, such as therapeutic methods. 1.
  • kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
  • the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, the term “about” indicates the designated value ⁇ one standard deviation of that value. [0030]
  • the term “combinations thereof” includes every possible combination of elements to which the term refers.
  • immunoglobulin refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically 1107368.00126 comprises three domains, CH1, CH2, and CH3.
  • Each light chain typically comprises a light chain variable region (V L ) and a light chain constant region.
  • the light chain constant region typically comprises one domain, abbreviated CL.
  • the term “Atypical chemokine receptor 4” or “ACKR4” describes a receptor for C-C type chemokines. Specifically, ACKR4 has been shown to bind dendritic cell and T cell-activated chemokines including CXCL13, CCL19, CCL20, CCL21, CCL22, and CCL25. ACKR4 has also been shown to act as a chemokine “scavenger” regulating the concentration of chemokines by internalizing and degrading bound chemokines through lysosomal degradation.
  • antibody describes a type of immunoglobulin molecule and is used herein in its broadest sense.
  • An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), and antibody fragments and antigen binding proteins.
  • Antibodies comprise at least one antigen-binding domain.
  • An antigen-binding domain is an antigen binding domain formed by a VH-VL dimer.
  • An “ACKR4 antibody,” “anti-ACKR4 antibody,” “ACKR4 Ab,” “ACKR4-specific antibody,” or “anti-ACKR4 Ab” is an antibody, as described herein, which binds specifically to the antigen ACKR4.
  • the VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved.
  • the more conserved regions are called framework regions (FRs).
  • Each V H and V L generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen binding, and confer antigen specificity and binding affinity to the antibody.
  • the light chain from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.
  • the heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. 1107368.00126 [0037]
  • the amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol.
  • Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR- H2, and CDR-H3 as identified by the Kabat and Chothia schemes.
  • residue numbering is provided using both the Kabat and Chothia numbering schemes.
  • the numbering scheme used for identification of a particular CDR herein is the Kabat/Chothia numbering scheme. Where the residues encompassed by these two numbering schemes diverge, the numbering scheme is specified as either Kabat or Chothia. Table 1. Residues in CDRs according to Kabat and Chothia numbering schemes.
  • an “antibody fragment” comprises a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody.
  • Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab′) 2 fragments, Fab′ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
  • “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain 1107368.00126 variable domain and one light chain variable domain.
  • “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments may be generated, for example, by papain digestion of a full-length antibody.
  • “F(ab′)2” fragments contain two Fab′ fragments joined, near the hinge region, by disulfide bonds. F(ab′) 2 fragments may be generated, for example, by pepsin digestion of an intact antibody.
  • the F(ab′) fragments can be dissociated, for example, by treatment with ß- mercaptoethanol.
  • Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a V H domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). Antibodies from Escherichia coli. In Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.
  • scFv-Fc fragments comprise an scFv attached to an Fc domain.
  • an Fc domain may be attached to the C-terminal of the scFv.
  • the Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ). Any suitable Fc domain known in the art or described herein may be used.
  • the term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies.
  • a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody.
  • a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. 1107368.00126 [0048] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.
  • a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated antibody is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator.
  • an isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain.
  • An isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment is not present.
  • an isolated antibody is prepared by at least one purification step. [0051] In some embodiments, an isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight.
  • an isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of an antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent. 1107368.00126 [0052] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ).
  • K D dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology, such as a Biacore ® instrument, or using bio-layer interferometry technology, such as an Octet ® instrument.
  • SPR surface plasmon resonance
  • Biacore ® instrument such as a Biacore ® instrument
  • bio-layer interferometry technology such as an Octet ® instrument.
  • the terms “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule.
  • Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target.
  • KD KD
  • an “affinity matured” antibody is one with one or more alterations in one or more CDRs or FRs that result in an improvement in the affinity of the antibody for its antigen, compared to a parent antibody which does not possess the alteration(s).
  • an affinity matured antibody has nanomolar or picomolar affinity for the target antigen.
  • Affinity matured antibodies may be produced using a variety of methods known in the art. For example, Marks et al.
  • the term “competes with” or “cross-competes with” indicates that the two or more antibodies compete for binding to an antigen (e.g., ACKR4).
  • ACKR4 is coated on a plate and allowed to bind a first antibody, after which a second, labeled antibody is added. If the presence of the first antibody reduces binding of the second antibody, then the antibodies compete.
  • the term “competes with” also includes combinations of antibodies where one antibody reduces binding of another antibody, but where no competition is observed when the antibodies are added in the reverse order.
  • the first and second antibodies inhibit binding of each other, regardless of the order in which they are added.
  • one antibody reduces binding of another antibody to its antigen by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • epitope means a portion of an antigen capable of specific binding to an antibody. Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding.
  • the epitope to which an antibody binds can be determined using known techniques for epitope determination such as, for example, testing for antibody binding to ACKR4 variants with different point-mutations.
  • Percent “identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software 1107368.00126 such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0062] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art.
  • Polypeptide sequences having such substitutions are known as “conservatively modified variants.”
  • the following groups of amino acids are considered conservative substitutions for one another.
  • amino acid refers to the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine
  • Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject.
  • “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject.
  • “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder.
  • the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or composition that when administered to a subject is effective to treat a disease or disorder.
  • the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, avians, goats and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an autoimmune disease or condition, and/or an infection that can be treated with an antibody provided herein.
  • the subject is a human that is suspected to have cancer, an autoimmune disease or condition, and/or an acute infection and chronic infection. 1107368.00126 2.
  • a first aspect provides an antibodies that selectively bind human ACKR4. In some embodiments, the antibody selectively binds to the extracellular domain of human ACKR4.
  • the antibody has one or more CDRs having particular lengths, in terms of the number of amino acid residues. In some embodiments, the Chothia CDR-H1 of the antibody is 6, 7, 8, or 9 residues in length. In some embodiments, the Kabat CDR-H1 of the antibody is 4, 5, 6, or 7 residues in length.
  • the Chothia CDR-H2 of the antibody is 5, 6, or 7 residues in length. In some embodiments, the Kabat CDR-H2 of the antibody is 15, 16, 17, or 18 residues in length. In some embodiments, the Kabat/Chothia CDR-H3 of the antibody is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 residues in length. [0070] In some embodiments, the Kabat/Chothia CDR-L1 of the antibody is 9, 10, 11, 12, 13, 14, 15, or 16 residues in length. In some embodiments, the Kabat/Chothia CDR-L2 of the antibody is 6, 7, or 8 residues in length.
  • the Kabat/Chothia CDR- L3 of the antibody is 8, 9, 10, 11, or 12 residues in length.
  • the antibody comprises a light chain.
  • the light chain is a kappa light chain.
  • the light chain is a lambda light chain.
  • the antibody comprises a heavy chain.
  • the heavy chain is an IgA.
  • the heavy chain is an IgD.
  • the heavy chain is an IgE.
  • the heavy chain is an IgG.
  • the heavy chain is an IgM.
  • the heavy chain is an IgG1.
  • the heavy chain is an IgG2. In some embodiments, the heavy chain is an IgG3. In some embodiments, the heavy chain is an IgG4. In some embodiments, the heavy chain is an IgA1. In some embodiments, the heavy chain is an IgA2. [0073] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab′)2 fragment. In some embodiments, the antibody fragment is a Fab′ fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment.
  • the antibody fragment is an scFv-Fc fragment.
  • the antibody is a monoclonal antibody. In some 1107368.00126 embodiments, the antibody is a polyclonal antibody. [0075] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. [0076] In some embodiments, the antibody is an affinity matured antibody. In some embodiments, the antibody is an affinity matured antibody derived from an illustrative sequence provided in this disclosure. [0077] In some embodiments, the antibody binds to ACKR4 and causes internalization of ACKR4.
  • the antibody binds to ACKR4 and causes degradation of ACKR4. In some embodiments, the antibody binds ACKR4 inhibiting the chemokine scavenging function of ACKR4.
  • the antibodies provided herein may be useful for the treatment of a variety of diseases and conditions, including cancers, autoimmune diseases, and infections. In some embodiments, the antibody inhibits ACKR4 chemokine scavenging. In some embodiments, the antibody internalizes and down-regulates ACKR4. [0079] In some embodiments, the antibody competes or is capable of competing for binding to human ACKR4 with another antibody or chemokine.
  • the antibody comprises or consists an antibody that is capable of competing for binding to human ACKR4 with a reference antibody, wherein the reference antibody binds to an epitope comprising position Cys262-Glu289, Met1-Val42, Tyr176-Gln201, and/or Met1-Lys41 of SEQ ID NO: 147 on a human ACKR4 polypeptide.
  • the antibody and the reference antibody cross-compete or are capable of cross-competing for binding to human ACKR4 with another antibody or chemokine.
  • the antibody binds to an epitope of SEQ ID NO: 147 on a human ACKR4 polypeptide.
  • the epitope comprises or consists of a contiguous or non-contiguous span of amino acids of the sequence set forth in SEQ ID NO: 147. In some embodiments, the epitope comprises a sequence that is identical or corresponds to a sequence that is within the sequence set forth in SEQ ID NO: 147. In some embodiments, the epitope has a sequence that has a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to a sequence that is within the sequence set forth in SEQ ID NO: 147. In some embodiments, the epitope has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions from a sequence that is within the sequence set forth in forth in SEQ ID NO: 147.
  • the epitope has 1, 2, or 3 1107368.00126 substitutions from residues a sequence that is within the sequence set forth in SEQ ID NO: 147.
  • the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48. In some embodiments, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 45. In some embodiments, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 46.
  • the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 47. In some embodiments, the antibody comprises a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 48. [0082] In some embodiments, the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure. In some embodiments, the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure.
  • the CDR- H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.
  • V H Sequences Comprising Illustrative CDRs [0083]
  • the antibody comprises a VH sequence comprising one or more CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-H sequences provided in this disclosure, and variants thereof.
  • the antibody comprises a V H sequence comprising one or more Kabat CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Kabat CDR-H sequences provided in this disclosure, and variants thereof.
  • 2.2.1.1.Kabat CDR-H3 the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 45. In some embodiments, the antibody comprises a VH sequence 1107368.00126 comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 46. In some embodiments, the antibody comprises a V H sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 47. In some embodiments, the antibody comprises a V H sequence comprising a Kabat CDR- H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 48. .
  • the antibody comprises a V H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 23-25. In some embodiments, the antibody comprises a V H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 23. In some embodiments, the antibody comprises a V H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 24.
  • the antibody comprises a V H sequence comprising a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 25. 2.2.1.3.Kabat CDR-H1 [0087] In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-5. In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2. In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 3. In some embodiments, the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 4. In some embodiments, the antibody comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 5.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48 and a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 23-25.
  • the Kabat CDR-H3 sequence and the Kabat CDR-H2 sequence are both from a single illustrative V H sequence provided in this disclosure.
  • the Kabat CDR-H3 and Kabat CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOS: 89-94. 2.2.1.5.Kabat CDR-H3 + Kabat CDR-H1
  • the antibody comprises a VH sequence comprising a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48, and a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-5.
  • the Kabat CDR-H3 sequence and the Kabat CDR-H1 sequence are both from a single illustrative V H sequence provided in this disclosure.
  • the Kabat CDR-H3 and Kabat CDR-H1 are both from a single illustrative VH sequence selected from SEQ ID NOS: 89-94.
  • the Kabat CDR-H1 and Kabat CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOS: 89-94. 2.2.1.7.Kabat CDR-H1 + Kabat CDR-H2 + Kabat CDR-H3 [0091]
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 1-5, a Kabat CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 23-25, and a Kabat CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48.
  • the Kabat CDR-H1 sequence, Kabat CDR-H2 sequence, and Kabat CDR-H3 sequence are all from a single illustrative VH sequence provided in this disclosure.
  • the Kabat CDR-H1, Kabat CDR-H2, and Kabat CDR-H3 are all from a single illustrative VH sequence selected from SEQ ID NOS: 89-94. 1107368.00126 [0092]
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 1, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45.
  • the antibody comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 2, a Kabat CDR-H2 sequence comprising SEQ ID NO: 24, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 46.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 3, a Kabat CDR-H2 sequence comprising SEQ ID NO: 25, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 47.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 4, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45.
  • the antibody comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 3, a Kabat CDR-H2 sequence comprising SEQ ID NO: 25, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 48.
  • the antibody comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 5, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45. 2.2.1.8.Variants of V H Sequences Comprising Illustrative Kabat CDRs [0093]
  • the V H sequences provided herein comprise a variant of an illustrative Kabat CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure.
  • the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H3 sequence provided in this disclosure.
  • the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H3 sequences provided in this disclosure.
  • the Kabat CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H2 sequence provided in this disclosure.
  • the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity 1107368.00126 with any of the illustrative Kabat CDR-H2 sequences provided in this disclosure.
  • the Kabat CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.
  • the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a variant of an illustrative Kabat CDR-H1 sequence provided in this disclosure.
  • the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Kabat CDR-H1 sequences provided in this disclosure.
  • the Kabat CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Kabat CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions. 2.2.2.
  • the antibody comprises a V H sequence comprising one or more Chothia CDR-H sequences comprising, consisting of, or consisting essentially of one or more illustrative Chothia CDR-H sequences provided in this disclosure, and variants thereof.
  • 2.2.2.1.Chothia CDR-H3 [0098]
  • the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 45. In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 46. In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 47. In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR- H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 48.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence 1107368.00126 selected from SEQ ID NOS: 34-36. In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 34. In some embodiments, the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 35.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 36. 2.2.2.3.Chothia CDR-H1 [00100] In some embodiments, the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 12-16. In some embodiments, the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 12.
  • the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 13. In some embodiments, the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 14. In some embodiments, the antibody comprises a V H sequence comprising a Chothia CDR- H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 15. In some embodiments, the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 16.
  • the antibody comprises a V H sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48, and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 34-36.
  • the Chothia CDR-H3 sequence and the Chothia CDR-H2 sequence are both from a single illustrative V H sequence provided in this disclosure.
  • the Chothia CDR-H3 and Chothia CDR-H2 are both from a single illustrative V H sequence selected from SEQ ID NOS: 89-94. 2.2.2.5.Chothia CDR-H3 + Chothia CDR-H1
  • the antibody comprises a VH sequence comprising a Chothia CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48, and a Chothia CDR-H1 sequence comprising, consisting 1107368.00126 of, or consisting essentially of a sequence selected from SEQ ID NOS: 12-16.
  • the Chothia CDR-H3 sequence and the Chothia CDR-H1 sequence are both from a single illustrative VH sequence provided in this disclosure.
  • the Chothia CDR-H3 and Chothia CDR-H1 are both from a single illustrative VH sequence selected from SEQ ID NOS: 89-94.
  • the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 12-16 and a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 34-36.
  • the Chothia CDR-H1 sequence and the Chothia CDR-H2 sequence are both from a single illustrative VH sequence provided in this disclosure.
  • the Chothia CDR-H1 and Chothia CDR-H2 are both from a single illustrative VH sequence selected from SEQ ID NOS: 89-94. 2.2.2.7.Chothia CDR-H1 + Chothia CDR-H2 + Chothia CDR-H3 [00104]
  • the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 12-16, a Chothia CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 34-36, and a Chothia CDR- H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 45-48.
  • the Chothia CDR-H1 sequence, Chothia CDR- H2 sequence, and Chothia CDR-H3 sequence are all from a single illustrative V H sequence provided in this disclosure.
  • the Chothia CDR-H1, Chothia CDR-H2, and Chothia CDR-H3 are all from a single illustrative V H sequence selected from SEQ ID NOS: 89-94.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 12, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45.
  • the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 13, a Chothia CDR-H2 sequence comprising SEQ ID NO: 35, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 46.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 14, a Chothia CDR-H2 sequence comprising SEQ ID NO: 36, and a Chothia CDR-H3 1107368.00126 sequence comprising SEQ ID NO: 47.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 15, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45.
  • the antibody comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 14, a Chothia CDR-H2 sequence comprising SEQ ID NO: 36, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 48.
  • the antibody comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 16, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45.
  • V H sequences provided herein comprise a variant of an illustrative Chothia CDR-H3, CDR-H2, and/or CDR-H1 sequence provided in this disclosure.
  • the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H3 sequence provided in this disclosure.
  • the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H3 sequences provided in this disclosure.
  • the Chothia CDR-H3 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a variant of an illustrative Chothia CDR-H2 sequence provided in this disclosure.
  • the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H2 sequences provided in this disclosure.
  • the Chothia CDR-H2 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the Chothia CDR-H1 sequence comprises, consists of, 1107368.00126 or consists essentially of a variant of an illustrative Chothia CDR-H1 sequence provided in this disclosure.
  • the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative Chothia CDR-H1 sequences provided in this disclosure.
  • the Chothia CDR-H1 sequence comprises, consists of, or consists essentially of any of the illustrative Chothia CDR-H1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.
  • V H Sequences [00110] In some embodiments, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 89-94. In some embodiments, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 89.
  • the antibody comprises a V H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 90. In some embodiments, the antibody comprises a V H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 91. In some embodiments, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 92. In some embodiments, the antibody comprises a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 93. In some embodiments, the antibody comprises a V H sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 94. 2.3.1.
  • the V H sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative VH sequence provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of a variant of an illustrative V H sequence provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any of the illustrative VH sequences provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of any of the illustrative V H sequences provided in this disclosure, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or 1107368.00126 fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions. 2.4.
  • the antibody comprises a VL sequence comprising one or more CDR-L sequences comprising, consisting of, or consisting essentially of one or more illustrative CDR-L sequences provided in this disclosure, and variants thereof.
  • CDR-L3 Sequences [00115] In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 77-81. In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 77.
  • the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 78. In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 79. In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 80. In some embodiments, the antibody comprises a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 81.
  • the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure.
  • the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure.
  • the CDR- L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. 2.5.1.
  • the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 67-70. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 67. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 68.
  • the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 69. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 70. 2.5.2. CDR-L1 [00118] In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 55-59.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 55. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 56. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 57. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 58.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 59. 2.5.3. CDR-L3 + CDR-L2 [00119] In some embodiments, the antibody comprises a VL sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 77-81 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 67-70. In some embodiments, the CDR- L3 sequence and the CDR-L2 sequence are both from a single illustrative VL sequence provided in this disclosure.
  • the CDR-L3 and CDR-L2 are both from a single illustrative VL sequence selected from SEQ ID NOS: 101-106. 2.5.4.
  • CDR-L3 + CDR-L1 [00120]
  • the antibody comprises a V L sequence comprising a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 77-81 and a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 55-59.
  • the CDR- L3 sequence and the CDR-L1 sequence are both from a single illustrative VL sequence provided in this disclosure.
  • the CDR-L3 and CDR-L1 are both from a single illustrative VL sequence selected from SEQ ID NOS: 101-106. 1107368.00126 2.5.5.
  • CDR-L1 + CDR-L2 [00121]
  • the antibody comprises a V L sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 55-59 and a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 67-70.
  • the CDR- L1 sequence and the CDR-L2 sequence are both from a single illustrative V L sequence provided in this disclosure.
  • the CDR-L1 and CDR-L2 are both from a single illustrative V L sequence selected from SEQ ID NOS: 101-106. 2.5.6.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 55-59, a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 67-70, and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 77-81.
  • the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all from a single illustrative VL sequence provided in this disclosure.
  • the CDR-L1, CDR-L2, and CDR-L3 are all from a single illustrative VL sequence selected from SEQ ID NOS: 101-106.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 67, and a CDR-L3 sequence SEQ ID NO: 77.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 56, a CDR-L2 sequence comprising SEQ ID NO: 68, and a CDR-L3 sequence SEQ ID NO: 78. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 69, and a CDR-L3 sequence SEQ ID NO: 79.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 77. In some embodiments, the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 58, a CDR- L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 80.
  • the antibody comprises a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81. 1107368.00126 2.5.7.
  • V L sequences comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81. 1107368.00126 2.5.7.
  • V L sequences comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81. 1107368.00126 2.5.7.
  • the CDR-L2 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L2 sequence provided in this disclosure. In some embodiments, the CDR-L2 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L2 sequences provided in this disclosure. In some embodiments, the CDR-L2 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L2 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.
  • the CDR-L1 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L1 sequence provided in this disclosure. In some embodiments, the CDR-L1 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L1 sequences provided in this disclosure. In some embodiments, the CDR-L1 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L1 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. 2.6.
  • the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 101-106. In some embodiments, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 101. In some embodiments, the antibody comprises a 1107368.00126 VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 102. In some embodiments, the antibody comprises a V L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 103. In some embodiments, the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 104.
  • the antibody comprises a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 105. In some embodiments, the antibody comprises a V L sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 106. 2.6.1. Variants of VL Sequences [00129] In some embodiments, the V L sequences provided herein comprise, consist of, or consist essentially of a variant of an illustrative VL sequence provided in this disclosure. [00130] In some embodiments, the VL sequence comprises, consists of, or consists essentially of a variant of an illustrative V L sequence provided in this disclosure.
  • the VL sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.05% identity with any of the illustrative VL sequences provided in this disclosure.
  • the VL sequence comprises, consists of, or consists essentially of any of the illustrative V L sequences provided in this disclosure, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions. 2.7. Pairs 2.7.1.
  • the antibody comprises a CDR-H3 sequence and a CDR- L3 sequence.
  • the CDR-H3 sequence is part of a VH and the CDR-L3 sequence is part of a V L .
  • the CDR-H3 sequence is a CDR-H3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 45-48 and the CDR-L3 sequence is a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 77-81.
  • the CDR-H3 sequence is SEQ ID NO: 45 and the CDR- L3 sequence is selected from SEQ ID NOS: 77-81.
  • the CDR-L3 1107368.00126 sequence is SEQ ID NO: 77.
  • the CDR-L3 sequence is SEQ ID NO: 78.
  • the CDR-L3 sequence is SEQ ID NO: 79.
  • the CDR-L3 sequence is SEQ ID NO: 80.
  • the CDR-L3 sequence is SEQ ID NO: 81.
  • the CDR-H3 sequence is SEQ ID NO: 46 and the CDR- L3 sequence is selected from SEQ ID NOS: 77-82.
  • the CDR-L3 sequence is SEQ ID NO: 77.
  • the CDR-L3 sequence is SEQ ID NO: 78.
  • the CDR-L3 sequence is SEQ ID NO: 79.
  • the CDR-L3 sequence is SEQ ID NO: 80.
  • the CDR-L3 sequence is SEQ ID NO: 81.
  • the CDR-H3 sequence is SEQ ID NO: 47 and the CDR- L3 sequence is selected from SEQ ID NOS: 77-82.
  • the CDR-L3 sequence is SEQ ID NO: 77.
  • the CDR-L3 sequence is SEQ ID NO: 78.
  • the CDR-L3 sequence is SEQ ID NO: 79.
  • the CDR-L3 sequence is SEQ ID NO: 80.
  • the CDR-L3 sequence is SEQ ID NO: 81.
  • the CDR-H3 sequence is SEQ ID NO: 48 and the CDR- L3 sequence is selected from SEQ ID NOS: 77-82.
  • the CDR-L3 sequence is SEQ ID NO: 77.
  • the CDR-L3 sequence is SEQ ID NO: 78.
  • the CDR-L3 sequence is SEQ ID NO: 79.
  • the CDR-L3 sequence is SEQ ID NO: 80.
  • the CDR-L3 sequence is SEQ ID NO: 81.
  • the CDR-H3 – CDR-L3 pairs provided herein comprise a variant of an illustrative CDR-H3 and/or CDR-L1 sequence provided in this disclosure.
  • the CDR-H3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-H3 sequence provided in this disclosure.
  • the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-H3 sequences provided in this disclosure.
  • the CDR- H3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-H3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some 1107368.00126 embodiments, the amino acid substitutions are conservative amino acid substitutions.
  • the CDR-L3 sequence comprises, consists of, or consists essentially of a variant of an illustrative CDR-L3 sequence provided in this disclosure. In some embodiments, the CDR-L3 sequence comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity with any of the illustrative CDR-L3 sequences provided in this disclosure. In some embodiments, the CDR-L3 sequence comprises, consists of, or consists essentially of any of the illustrative CDR-L3 sequences provided in this disclosure, with 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. 2.7.2.
  • the antibody comprises a VH sequence and a VL sequence.
  • the VH sequence is a VH sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 89-94 and the VL sequence is a VL sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 101-106.
  • the VH sequence is SEQ ID NO: 89 and the VL sequence is selected from SEQ ID NOS: 101-106.
  • the V L sequence is SEQ ID NO: 101.
  • the VL sequence is SEQ ID NO: 102.
  • the VL sequence is SEQ ID NO: 103. In some embodiments, the VL sequence is SEQ ID NO: 104. In some embodiments, the VL sequence is SEQ ID NO: 105. In some embodiments, the VL sequence is SEQ ID NO: 106. [00144] In some embodiments, the V H sequence is SEQ ID NO: 90 and the V L sequence is selected from SEQ ID NOS: 101-106. In some embodiments, the VL sequence is SEQ ID NO: 101. In some embodiments, the VL sequence is SEQ ID NO: 102. In some embodiments, the VL sequence is SEQ ID NO: 103. In some embodiments, the VL sequence is SEQ ID NO: 104.
  • the VL sequence is SEQ ID NO: 105. In some embodiments, the V L sequence is SEQ ID NO: 106. [00145] In some embodiments, the VH sequence is SEQ ID NO: 91 and the VL sequence is selected from SEQ ID NOS: 101-106. In some embodiments, the V L sequence is SEQ ID NO: 101. In some embodiments, the VL sequence is SEQ ID NO: 102. In some embodiments, the VL sequence is SEQ ID NO: 103. In some embodiments, the VL sequence is SEQ ID NO: 104. In some embodiments, the V L sequence is SEQ ID NO: 105. In some embodiments, the 1107368.00126 VL sequence is SEQ ID NO: 106.
  • the VH sequence is SEQ ID NO: 92 and the VL sequence is selected from SEQ ID NOS: 101-106.
  • the VL sequence is SEQ ID NO: 101.
  • the VL sequence is SEQ ID NO: 102.
  • the V L sequence is SEQ ID NO: 103.
  • the V L sequence is SEQ ID NO: 104.
  • the VL sequence is SEQ ID NO: 105.
  • the V L sequence is SEQ ID NO: 106.
  • the VH sequence is SEQ ID NO: 93 and the VL sequence is selected from SEQ ID NOS: 101-106.
  • the VL sequence is SEQ ID NO: 101. In some embodiments, the V L sequence is SEQ ID NO: 102. In some embodiments, the VL sequence is SEQ ID NO: 103. In some embodiments, the VL sequence is SEQ ID NO: 104. In some embodiments, the V L sequence is SEQ ID NO: 105. In some embodiments, the VL sequence is SEQ ID NO: 106. [00148] In some embodiments, the VH sequence is SEQ ID NO: 94 and the VL sequence is selected from SEQ ID NOS: 101-106. In some embodiments, the V L sequence is SEQ ID NO: 101. In some embodiments, the VL sequence is SEQ ID NO: 102.
  • the V L sequence is SEQ ID NO: 103. In some embodiments, the V L sequence is SEQ ID NO: 104. In some embodiments, the VL sequence is SEQ ID NO: 105. In some embodiments, the V L sequence is SEQ ID NO: 106. 2.7.3.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 1, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 67, and a CDR-L3 sequence SEQ ID NO: 77.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 2, a Kabat CDR-H2 sequence comprising SEQ ID NO: 24, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 46 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 56, a CDR-L2 sequence comprising SEQ ID NO: 68, and a CDR-L3 sequence SEQ ID NO: 78.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID 1107368.00126 NO: 3, a Kabat CDR-H2 sequence comprising SEQ ID NO: 25, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 47 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 69, and a CDR-L3 sequence SEQ ID NO: 79.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 4, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 77.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 3, a Kabat CDR-H2 sequence comprising SEQ ID NO: 25, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 48 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 69, and a CDR-L3 sequence SEQ ID NO: 79.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 4, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 58, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 80.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 5, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Kabat CDR-H1 sequence comprising SEQ ID NO: 4, a Kabat CDR-H2 sequence comprising SEQ ID NO: 23, and a Kabat CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 12, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 1107368.00126 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 67, and a CDR-L3 sequence SEQ ID NO: 77.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 13, a Chothia CDR-H2 sequence comprising SEQ ID NO: 35, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 46 and a VL sequence comprising a CDR- L1 sequence comprising SEQ ID NO: 56, a CDR-L2 sequence comprising SEQ ID NO: 68, and a CDR-L3 sequence SEQ ID NO: 78.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 14, a Chothia CDR-H2 sequence comprising SEQ ID NO: 36, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 47 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 69, and a CDR-L3 sequence SEQ ID NO: 79.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 15, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 55, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 77.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 14, a Chothia CDR-H2 sequence comprising SEQ ID NO: 36, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 48 and a VL sequence comprising a CDR- L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 69, and a CDR-L3 sequence SEQ ID NO: 79.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 15, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 58, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 80.
  • the binding domain capable of binding to an ACKR4 epitope comprises a V H sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 16, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR- 1107368.00126 L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81.
  • the binding domain capable of binding to an ACKR4 epitope comprises a VH sequence comprising a Chothia CDR-H1 sequence comprising SEQ ID NO: 16, a Chothia CDR-H2 sequence comprising SEQ ID NO: 34, and a Chothia CDR-H3 sequence comprising SEQ ID NO: 45 and a VL sequence comprising a CDR-L1 sequence comprising SEQ ID NO: 59, a CDR-L2 sequence comprising SEQ ID NO: 70, and a CDR-L3 sequence SEQ ID NO: 81.
  • the V H – V L pairs provided herein comprise a variant of an illustrative VH and/or VL sequence provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of a variant of an illustrative V H sequence provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.1% identity with any of the illustrative VH sequences provided in this disclosure.
  • the VH sequence comprises, consists of, or consists essentially of any of the illustrative V H sequences provided in this disclosure, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the V L sequence comprises, consists of, or consists essentially of a variant of an illustrative VL sequence provided in this disclosure. In some embodiments, the V L sequence comprises, consists of, or consists essentially of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.05% identity with any of the illustrative V L sequences provided in this disclosure.
  • the V L sequence comprises, consists of, or consists essentially of any of the illustrative VL sequences provided in this disclosure, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acid substitutions.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibody comprises or consists of one or more heavy chains consisting of an HC sequence and one or more light chains consisting of an LC sequence. In some embodiments, the antibody comprises or consists of two identical heavy chains consisting of an HC sequence and two identical light chains consisting of an LC sequence.
  • the HC sequence is an HC sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOS: 113-120 and the LC sequence is an LC sequence comprising, consisting of, or consisting essentially of SEQ ID NOS: 128-133.
  • the HC sequence is an HC sequence consisting of a sequence selected from SEQ ID NOS: 113-120 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 113 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-133.
  • the LC sequence is SEQ ID NO: 128.
  • the LC sequence is SEQ ID NO: 129.
  • the LC sequence is SEQ ID NO: 130.
  • the LC sequence is SEQ ID NO: 131.
  • the LC sequence is SEQ ID NO: 132. In some embodiments, the LC sequence is SEQ ID NO: 133. [00161] In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 114 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-134. In some embodiments, the LC sequence is SEQ ID NO: 128. In some embodiments, the LC sequence is SEQ ID NO: 129. In some embodiments, the LC sequence is SEQ ID NO: 130. In some embodiments, the LC sequence is SEQ ID NO: 131. In some embodiments, the LC sequence is SEQ ID NO: 132.
  • the LC sequence is SEQ ID NO: 133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 115 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-134.
  • the LC sequence is SEQ ID NO: 128.
  • the LC sequence is SEQ ID NO: 129.
  • the LC sequence is SEQ ID NO: 130.
  • the LC sequence is SEQ ID NO: 131.
  • the LC sequence is SEQ ID NO: 132.
  • the LC sequence 1107368.00126 is SEQ ID NO: 133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 116 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-134.
  • the LC sequence is SEQ ID NO: 128.
  • the LC sequence is SEQ ID NO: 129.
  • the LC sequence is SEQ ID NO: 130.
  • the LC sequence is SEQ ID NO: 131.
  • the LC sequence is SEQ ID NO: 132.
  • the LC sequence is SEQ ID NO: 133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 119 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-134.
  • the LC sequence is SEQ ID NO: 128.
  • the LC sequence is SEQ ID NO: 129.
  • the LC sequence is SEQ ID NO: 130.
  • the LC sequence is SEQ ID NO: 131.
  • the LC sequence is SEQ ID NO: 132.
  • the LC sequence is SEQ ID NO: 133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 120 and the LC sequence is an LC sequence consisting of a sequence selected from SEQ ID NOS: 128-134.
  • the LC sequence is SEQ ID NO: 128.
  • the LC sequence is SEQ ID NO: 129.
  • the LC sequence is SEQ ID NO: 130.
  • the LC sequence is SEQ ID NO: 131.
  • the LC sequence is SEQ ID NO: 132.
  • the LC sequence is SEQ ID NO: 133.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 113 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 128. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 114 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 129. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 115 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 130. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 116 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 131.
  • the HC sequence is an HC sequence consisting of SEQ ID NO: 117 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 130. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 118 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 132. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 119 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 133. In some embodiments, the HC sequence is an HC sequence consisting of SEQ ID NO: 124 and the LC sequence is an LC sequence consisting of sequence SEQ ID NO: 133. 2.8.
  • anti-ACKR antibodies comprising one or more sequences defined by consensus sequences.
  • Each consensus sequence is based, at least in part, on one or more alignments of two or more useful anti-ACKR4 CDR sequences provided in this disclosure. Based on such alignments, a person of skill in the art would recognize that different amino acid residues may useful in certain positions of the CDRs. Accordingly, each consensus sequence encompasses two or more useful anti-ACKR4 CDR sequences. 2.8.1.
  • the antibody comprises a CDR-H3 sequence defined by the consensus sequence D-I-I-T-H-Y-Y- ⁇ 8-Y-G-M-D-V (SEQ ID NOS: 47-48), where ⁇ 8 is Y or T. In some embodiments, ⁇ 8 is T. In some embodiments, ⁇ 8 is Y. 1107368.00126 2.8.2.
  • the antibody comprises a Chothia CDR-H2 sequence defined by the consensus sequence ⁇ 1- ⁇ 2- ⁇ 3-G- ⁇ 5- ⁇ 6 (SEQ ID NOS: 34-35), where ⁇ 1 is S or W; ⁇ 2 is G or Y; ⁇ 3 is G or D; ⁇ 5 is G or S; and ⁇ 6 is S or N.
  • SEQ ID NOS: 34-35 consensus sequence
  • ⁇ 1 is S
  • ⁇ 2 is G or Y
  • ⁇ 3 is G or D
  • ⁇ 5 is G or S
  • ⁇ 6 is S or N.
  • the antibody comprises a Chothia CDR-H1 sequence defined by the consensus sequence G-F-T-F-S- ⁇ 6- ⁇ 7 (SEQ ID NOS: 12-13 and 15-16), where ⁇ 6 is T, S or Y and ⁇ 7 is C, Y, or S.
  • ⁇ 6 is T; ⁇ 7 is C, or S.
  • ⁇ 6 is T or Y.
  • the antibody comprises a Kabat CDR-H1 sequence defined by the consensus sequence ⁇ 1- ⁇ 2-A-M-S (SEQ ID NOS: 1 and 4-5), where ⁇ 1 is T or Y; and ⁇ 2 is C or S.
  • ⁇ 1 when ⁇ 1 is T; ⁇ 2 is C or S. In some embodiments, when ⁇ 2 is S; ⁇ 1 is T or Y. [00181] In some embodiments, when ⁇ 1 is T; ⁇ 2 is C. In some embodiments, when ⁇ 1 is T; ⁇ 2 is S. In some embodiments, when ⁇ 1 is Y; ⁇ 1 is S. 1107368.00126 2.8.6.
  • the antibody comprises a CDR-L3 sequence defined by the consensus sequence ⁇ 1- Q- ⁇ 3- ⁇ 4- ⁇ 5-W-P- ⁇ 8-T (SEQ ID NOS: 77-78 and 80-81), where ⁇ 1 is Q or M; ⁇ 3 is R or G; ⁇ 4 is T, S, or A; ⁇ 5 .is N, H, or R; and ⁇ 8 is P or F.
  • ⁇ 1 is Q
  • ⁇ 3 is R
  • ⁇ 4 is S or A
  • ⁇ 8 is P.
  • ⁇ 4 when ⁇ 4 is S; ⁇ 1 is Q; ⁇ 3 is R; ⁇ 5.is N or R; and ⁇ 8 is P.
  • ⁇ 5 when ⁇ 1 is Q; ⁇ 3 is R; ⁇ 4 .is S or A; and ⁇ 8 is P.
  • ⁇ 1 when ⁇ 1 is Q; ⁇ 3 is R; ⁇ 4 is S; ⁇ 5.is N; and ⁇ 8 is P.
  • ⁇ 1 is M; ⁇ 3 is G; ⁇ 4 is T; ⁇ 5.is H; and ⁇ 8 is F.
  • ⁇ 1 when ⁇ 1 is Q; ⁇ 3 is R; ⁇ 4 is S; ⁇ 5 .is R; and ⁇ 8 is P. In some embodiments, when ⁇ 1 is Q; ⁇ 3 is R; ⁇ 4 is A; ⁇ 5.is R; and ⁇ 8 is P. 2.8.7.
  • the antibody comprises a CDR-L2 sequence defined by the consensus sequence ⁇ 1- ⁇ 2- ⁇ 3-N-R- ⁇ 6- ⁇ 7 (SEQ ID NOS: 67-70), where ⁇ 1 is D, R, or A; ⁇ 2 is V or A; ⁇ 3 is N or S; ⁇ 6 is A, D or P; and ⁇ 7 is T or S.
  • SEQ ID NOS: 67-70 consensus sequence
  • ⁇ 1 is D, R, or A
  • ⁇ 2 is V or A
  • ⁇ 3 is N or S
  • ⁇ 6 is A, D or P
  • ⁇ 7 is T or S.
  • ⁇ 1 when ⁇ 1 is R; ⁇ 2 is V; ⁇ 3 is S; ⁇ 6 is D; and ⁇ 7 is S.
  • ⁇ 1 when ⁇ 1 is A; ⁇ 2 is V; ⁇ 3 is N; ⁇ 6 is P; and ⁇ 7 is S.
  • ⁇ 1 when ⁇ 1 is D; ⁇ 2 is V; ⁇ 3 is S; ⁇ 6 is A; and ⁇ 7 is T. 2.8.8.
  • the antibody comprises a CDR-L1 sequence defined by the consensus sequence R-A-A-Q-S-V- ⁇ 7- ⁇ 8-S-L-A (SEQ ID NOS: 55 and 58-59), where ⁇ 7 is S or R; and ⁇ 8 is S or R.
  • ⁇ 7 is S
  • ⁇ 8 is S or R
  • when ⁇ 8 is R; ⁇ 7 is S or R.
  • the antibody decreases the affinity of ACKR4 for its chemokines. In some embodiments, the antibody disrupts the association of ACKR4 with CCL19, CCL21, CCL25, and/or other C-C type chemokines. In some embodiments the antibody prevents ACKR4 internalization of chemokines. In some embodiments the antibody prevents ACKR4 degradation of chemokines. In some embodiments the antibody inhibits chemokine scavenging by ACKR4. [00192] In some embodiments, the antibody increases internalization of ACKR4.
  • the antibody down-regulates ACKR4.
  • the decrease is about or less than a 10% decrease, about or less than a 20% decrease, about or less than a 30% decrease, about or less than a 40% decrease, about or less than a 50% decrease, about or less than a 60% decrease, about or less than a 70% decrease, about or less than an 80% decrease, about or less than a 90% decrease, or about a complete decrease.
  • the increase is about or greater than a 10% increase, about or greater than a 20% increase, about or greater than a 30% increase, about or greater than a 40% increase, about or greater than a 50% increase, about or greater than a 60% increase, about or greater than a 70% increase, about or greater than an 80% increase, about or greater than a 90% increase, or a complete increase.
  • ACKR4 Assays [00194]
  • the antibody binds to an epitope of ACKR4.
  • An epitope often consists of a number of contiguous amino acids such as, for example, without limitation, 5-6 amino acids.
  • the epitope comprises or consists of contiguous or non-contiguous amino acids.
  • the contiguous or non-contiguous amino acids are within a domain of ACKR4.
  • ACKR4 has a sequence identical to the amino acid sequence set forth in SEQ ID NO: 147.
  • the epitope has an amino acid sequence that is within the amino acid sequence set forth in SEQ ID NO: 147.
  • the epitope has an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to a sequence that is within the sequence set forth in SEQ ID NO: 147.
  • the epitope has a sequence that has a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to a sequence that is within the sequence set forth in SEQ ID NO: 147. In some embodiments, the epitope has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions 1107368.00126 from a sequence that is within the sequence set forth in forth in SEQ ID NO: 147. In some embodiments, the epitope has 1, 2 or 3 substitutions from residues a sequence that is within the sequence set forth in SEQ ID NO: 147. [00197] In some embodiments, the antibody competes with any of the antibodies set forth herein.
  • glycosylation site refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • Addition or deletion of N-linked glycosylation sites to the antibody may be accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences is created or removed.
  • Addition or deletion of O-linked glycosylation sites may be accomplished by addition, deletion, or substitution of one or more serine or threonine residues in or to (as the case may be) the sequence of an antibody.
  • the antibody is glycosylated.
  • the antibody is deglycosylated. Carbohydrates may be removed by standard techniques.
  • the antibody is aglycosylated, for instance by expression in a system that does not glycosylate. 6.
  • Fc Variants [00203]
  • amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant.
  • the Fc region variant possesses some, but not all, effector functions.
  • an IgG Fc is engineered to modulate antibody effector function (See Wang et al., Protein Cell, 2018, Jan; 9(1): 63–73), which is incorporated by reference herein in its entirety, including any drawings).
  • in vitro assays to assess ADCC activity of a molecule of interest are provided in U.S. Patent Nos. 5,500,362 and 5,821,337; Hellstrom et al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83:7059-7063; Hellstrom et al., Proc. Natl. Acad. Sci.
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity.
  • C1q binding assays examples include those described in WO 2006/029879 and WO 2005/100402.
  • Complement activation assays include those described, for example, in Gazzano-Santoro et al., J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003, 101:1045-1052; and Cragg and Glennie, Blood, 2004, 103:2738-2743.
  • FcRn binding and in vivo clearance can also be measured, for example, using the methods described in Petkova et al., Intl. Immunol., 2006, 18:1759-1769. 7.
  • Monoclonal antibodies may also be obtained, for example, using phage or yeast-based libraries. See e.g., U.S. Patent Nos. 8,258,082 and 8,691,730.
  • a mouse or other appropriate host animal is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • Useful myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive media conditions, such as the presence or absence of HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, MD).
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. See e.g., Kozbor, J.
  • hybridoma cells that produce antibodies of the desired specificity, affinity, and/or biological activity
  • selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • DNA encoding the monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells can serve as a useful source of DNA encoding antibodies with the desired properties.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as bacteria (e.g., E.
  • Humanized antibodies may be generated by replacing most, or all, of the structural portions of a monoclonal antibody with corresponding human antibody sequences. Consequently, a hybrid molecule is generated in which only the antigen-specific variable, or CDR, is composed of non-human sequence. Methods to obtain humanized antibodies include those described in, for example, Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci.
  • Human antibodies can be generated by a variety of techniques known in the art, for example by using transgenic animals (e.g., humanized mice). See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
  • Human antibodies can also be derived from phage-display libraries (see e.g., Hoogenboom et al., J. Mol. Biol., 1991, 227:381-388; Marks et al., J. Mol. Biol., 1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905).
  • the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression.
  • the nucleic acid may be produced by homologous recombination, for example as described in U.S. Patent No.5,204,244.
  • Many different vectors are known in the art.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Patent No.5,534,615.
  • Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P.
  • E. coli Escherichia
  • Enterobacter Erwinia
  • Klebsiella Proteus
  • Salmonella S. typhimurium
  • Serratia S. marcescans
  • Shigella Bacilli (B. subtilis and B. licheniformis
  • E. coli 294 One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-ACKR4 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is a commonly used lower eukaryotic host microorganism.
  • Schizosaccharomyces pombe Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as, for example Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.
  • the particulate debris either host cells or lysed fragments
  • the particulate debris is removed, for example, by centrifugation or ultrafiltration.
  • Carter et al. Bio/Technology, 1992, 10:163-167 describes a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes.
  • PMSF phenylmethylsulfonylfluoride
  • the antibody is produced in a cell-free system.
  • the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs, 2012, 4:217-225, incorporated by reference in its entirety.
  • the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell.
  • the prokaryotic cell is E. coli.
  • Cell-free expression of the antibody may be useful, for example, where the antibody accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.
  • the pharmaceutical composition comprises an anti- foaming agent. Any suitable anti-foaming agent may be used.
  • the anti- foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof.
  • the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.
  • the pharmaceutical composition comprises a surfactant.
  • surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.
  • the pharmaceutical composition comprises an anti- caking agent.
  • anti-caking agents include calcium phosphate (tribasic), 1107368.00126 hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.
  • Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars.
  • compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable 1107368.00126 formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
  • Excipients that increase the solubility of one or more of the antibodies disclosed herein can also be incorporated into the parenteral dosage forms. 9.2. Dosage and Unit Dosage Forms [00252] In human therapeutics, the doctor will determine the dosology which she considers most appropriate according to a preventive or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated. [00253] The amount of the antibody or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the antibody is administered.
  • the frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • exemplary doses of a composition include milligram or microgram amounts of the antibody per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram).
  • the dosage of the antibody provided herein, based on weight of the antibody, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject’s body weight.
  • the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1107368.00126 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.25 mg to 2.5 mg, 0.5 mg to 20 mg, 0.5 to 15 mg, 0.5 to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1
  • the dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the antibody outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response. [00256] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the antibodies provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules.
  • a subject when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same.
  • the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.
  • treatment or prevention can be initiated with one or more loading doses of an antibody or composition provided herein followed by one or more maintenance doses.
  • a dose of an antibody or composition provided herein can be administered to achieve a steady-state concentration of the antibody in blood or serum of the subject.
  • the steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight and age.
  • administration of the same composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
  • administration of the same prophylactic or therapeutic agent may be repeated and the 1107368.00126 administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. 10.
  • the antibodies also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • the intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.
  • the antibodies provided herein may be useful for the treatment of any disease or condition, such as cancer, autoimmune disease, and infection.
  • Any suitable cancer may be treated with the antibodies provided herein.
  • Illustrative suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbla
  • the cancer is selected from breast, lung, CRC, gastric, esophageal, neuroblastoma, cervical, and hematological cancers.
  • Any suitable autoimmune disease may be treated with the antibodies provided herein.
  • Illustrative suitable autoimmune diseases, or diseases with an autoimmune component include, for example, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis
  • AIED autoimmune
  • the bispecific antibody or the complexing antibody is administered in an amount sufficient to achieve 1, 2, 3, 4, 5, 6, 7, or 8 of the following in the subject: a) inhibition of immune suppression; b) reduction of levels of regulatory T cells; c) increase an activity of myeloid cells; d) increase in activity of cytotoxic T lymphocytes, NK cells, B cells, neutrophils, monocytes, macrophages, and/or dendritic cells; e) increase in phagocytic activity; f) inhibition of metastasis; g) inhibition of tumor growth; and/or h) induction of tumor regression.
  • the bispecific antibody binds ACKR4 and a target protein.
  • the method for modulating immune system function in 1107368.00126 a subject in need thereof further comprises administering chemotherapy, administering radiation therapy, and/or administering one or more additional therapeutic agents.
  • the one or more additional therapeutic agents comprise one or more immunostimulatory agents.
  • the one or more immunostimulatory agents comprise an antagonist to an inhibitory receptor of an immune cell.
  • the one or more immunostimulatory agents comprise an agonist of a co-stimulatory receptor of an immune cell.
  • the co- stimulatory receptor comprises one or more of OX40, CD2, CD27, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp30, NKp46, NKp80, CD160, and/or a CD83 ligand.
  • the one or more immunostimulatory agents comprise a cytokine.
  • the cytokine is at least one of IL-1, IL-2, IL-5, IL-7, IL- 10, IL-12, IL-15, IL-21, and/or IL-27.
  • the one or more immunostimulatory agents comprise an oncolytic virus.
  • the oncolytic virus comprises one or more of the oncolytic virus is a Herpes simplex virus, a Vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, or a maraba virus.
  • the one or more immunostimulatory agents comprise a chimeric antigen engineered T cell.
  • immunostimulatory agents comprise a bi- or multi- specific T cell directed antibody.
  • the one or more immunostimulatory agents comprises or consists of an ADCC competent antibody that may target CD19, CD20, EGFR, Her2, SLAMF7, CD52, BCMA, GD2, CD38, or CCR4.
  • the ADCC competent antibody is effector enhanced through afucosylation, point mutations, or otherwise.
  • the one or more immunostimulatory agents comprise a bi-specific T cell engager and/or CAR-T therapy, CAR-NK therapy, CAR-macrophage therapy, adoptive T cell therapy. 11.
  • kits 1107368.00126 an anti-ACKR4 antibody provided herein is provided in the form of a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure.
  • the procedure is a diagnostic assay.
  • the procedure is a therapeutic procedure.
  • the kit further comprises a solvent for the reconstitution of the anti-aCKR antibody.
  • the anti-ACKR4 antibody is provided in the form of a pharmaceutical composition.
  • ACKR4 antibodies were discovered by the hybridoma method as first described by Kohler et al, Nature, 1975, 256:495-497 (incorporated herein by reference in its entirety) or a B cell data mining approach as provided below. The sequences and characteristics of the ACKR4 binding antibodies from these methods are provided in Table S and FIG.1. [00276] Since ACKR4 is a GPCR and reliable recombinant protein is not readily available, a genetic immunization strategy using cDNA was selected.
  • the hACKR4 cDNA was either full length, truncated at the N-terminus by 15 amino acids, or truncated at the N-terminus by 32 amino acids.
  • the truncated cDNA constructs were designed to focus immune responses on the membrane-proximal region of the N-terminal domain (heretofore “NTD”) and the extracellular loops (heretofore “ECLs”).
  • NTD membrane-proximal region of the N-terminal domain
  • ECLs extracellular loops
  • Transgenic rats as described in Ma et al, J. Immunol. Methods, 2013, 400-401:78-86 (incorporated herein by reference in its entirety), “OmniRats,” were immunized with one of the three cDNA constructs and immune responses were monitored via test bleeds at day 45 post-immunization initiation.
  • the seropositive animals were distributed to antibody recovery by either hybridoma or B cell sequencing methods.
  • Six animals were selected for a single hybridoma fusion and supernatants were screened by both flow cytometry and cell ELISA against cell lines engineered to overexpress ACKR4 to identify clones with the strongest binding to ACKR4 with minimal background binding to cells that did not express ACKR4. Top clones derived from hybridomas were sequenced by the Sanger sequencing method.
  • VH/VL sequences were paired based on the initial cell barcoding.
  • Over 400 fully human IgG1 antibodies were expressed in HEK293 cells and screened as crude cell culture supernatants by both flow cytometry and cell ELISA.36 unique ACKR4-binding clones were identified for cloning into recombinant IgG vectors for production, purification, and screening.
  • Purified antibodies with the lowest EC50 for ACKR4 i.e. best binders
  • Three antagonist clones were selected as leads and advanced to sequence optimization to remove sequence liabilities.
  • Example 2 Affinity Maturation of ACKR4 Antibodies
  • VLPs virus lipid particles
  • All six CDRs were diversified in the affinity maturation library designs. Specific residues within each CDR were varied only to amino acids that were present in known human IgGs from each germline. Total combined heavy and light chain diversity for each lineage was greater than 1E9 variants.
  • ACKR4 IgGs and recombinant human ACKR4 full length micelles (Ala2- Ile350) (ACKR4-FL-FLAG-Avi) were expressed and purified from Expi293 cells using transient transfection with ExpiFectamine (Thermo Fisher Scientific). Enhancers were added 20 h after transfection.
  • ACKR4 micelles medium was removed by centrifugation at 4,000g for 20 min and the Expi293 cell pellet was lysed on ice in 10 mM Tris-HCl pH 7.4, 1 mM EDTA with protease inhibitors (Roche) using a dounce tissue grinder (Kimble), centrifuged at 24,000g for 45 min at 4 °C to remove the supernatant containing cytoplasmic proteins and isolate the membrane pellet containing ACKR4.
  • the membrane pellet was solubilized in 67 mM HEPES pH 7.4, 533 mM sodium chloride (NaCl), 1% n-Dodecyl- ⁇ -D-Maltopyranoside (DDM) (Anatrace), and 0.2% Cholesteryl Hemisuccinate Tris Salt (CHS) (Anatrace) using a dounce tissue grinder, centrifuged at 24,000g for 90 min at 4 °C to isolate the supernatant containing ACKR4, and purified by ANTI-FLAG M2 Affinity Gel (Millipore Sigma).
  • ACKR4-FL- FLAG-Avi was further purified using SEC (Cytiva) in micelle buffer (30 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% DDM, and 0.02% CHS), concentrated, and flash frozen for storage at -80 °C. [00285] Purity and integrity of all proteins were assessed by HPLC and SDS-PAGE.
  • Example 4 Cell Lines [00286] Cell lines were grown and maintained in T75 flasks at 37°C and 5% CO2.
  • CHO cells were grown in F12K supplemented with 10% fetal bovine serum (FBS), 1% penicillin streptomycin (Pen Strep), 1% HEPES, 1% MEM non-essential amino acids (MEM NEAA), and 1% sodium pyruvate.
  • FBS fetal bovine serum
  • Pen Strep penicillin streptomycin
  • HEPES 1% HEPES
  • sodium pyruvate MC38-hACKR4, MC38-parental, SUIT2 WT, SUIT2 ACKR4 KO, AsPC-1 WT, and AsPC-1 ACKR4 KO were grown in RPMI supplemented with 10% FBS, 1% Pen Strep, 1% HEPES, 1% MEM NEAA, and 1% sodium pyruvate.
  • Example 5 Monovalent Affinity of ACKR4 Antibody Binding to Recombinant ACKR4 1107368.00126 Micelles
  • Binding kinetics were measured using the Octet RED96 system (ForteBio) at 30° C in micelle buffer. 2 ug/ml of ACKR4 antibody or control was immobilized onto anti- human Fc (AHC) biosensor to a binding response of approximately 0.5 nm. After a short baseline step in micelle buffer, the sensors were exposed to 200 nM ACKR4-FL-FLAG-Avi for the association step. Dissociation of the complex was monitored upon exposure of the sensors to micelle buffer for up to 10 min.
  • BSA Stain Buffer
  • ACKR4 antibody or control washed with 0.02-400 nM (4-fold dilutions) ACKR4 antibody or control for 30 min at 4 °C.
  • Cells were washed with Stain Buffer (BSA), fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) for 30 min at 4 °C.
  • Cells were washed with Stain Buffer (BSA) and incubated with anti-human IgG R-Phycoerythrin (R-PE) secondary antibody (Jackson ImmunoResearch) for 30 min at 4°C.
  • BSA Stain Buffer
  • R-PE anti-human IgG R-Phycoerythrin
  • FIG. 1 provides a table showing CHO-hACKR4 binding by the ACKR4 antibodies. 1107368.00126 [00292]
  • FIG.2A shows CHO-hACKR4 binding by representative ACKR4 antibodies.
  • Example 7 Epitope Mapping of ACKR4 Antibodies
  • CHO-parental, CHO-hACKR4, CHO-mACKR4, ECL2 mutant (CHO- mACKR4 cells with mouse to human ECL2 mutations Q180D / T185I / H190R / S199L), and ECL3 mutant (CHO-mACKR4 cells with mouse to human ECL3 mutations Q263R / A267I / L270S / D276N) cells were detached with Accutase and washed with 1X PBS.0.05-0.1 million cells were stained with Fixable Viability Dye eFluorTM 780 (Thermo Fisher Scientific) for 30 min at 4°C.
  • FIG. 3 shows CHO ACKR4 and ECL mutant cell binding by ACKR4 antibodies. Some antibodies disclosed herein are cross-reactive binders that do not bind to ECL2 and ECL3; some antibodies bind to ECL3.
  • Example 8 Inhibition of Chemokine Scavenging by ACKR4 Antibodies in Endogenous ACKR4 Cells
  • Inhibition of chemokine scavenging assays were performed in polypropylene plates rather than polystyrene plates due to high non-specific binding of CCL19 and CCL21 to the polystyrene plates.
  • Flat bottom polypropylene plates (Corning) were coated with 10 ug/ml human fibronectin (PromoCell) for 1 h at 37 °C and blocked with bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • FIG. 4A and 4B show inhibition of CCL21 scavenging by representative ACKR4 antibodies in AsPC-1 WT and SUIT2 WT cells, respectively.
  • FIG. 4C shows inhibition of CCL19 scavenging by representative ACKR4 antibodies in SUIT2 WT cells.
  • Example 9 HiBiT Detection of ACKR4 Internalization and Down-regulation by ACKR4 Antibodies in ACKR4 Overexpressing Cells [00298] 0.05 million CHO-HiBiT-ACKR4 cells were treated with 100 ug/ml ACKR4 antibody or control in white flat bottom plate (Corning) for 4-48 h at 37° C.
  • FIG. 5A shows extracellular luminescence of HiBiT-ACKR4 upon treatment with ACKR4 antibody or control after 15 h.
  • FIG.5B shows % HiBiT-ACKR4 internalization by ACKR4 antibody calculated by: 100 X [1 - (RLU of treatment / RLU of Neg Ctl IgG)].
  • FIG. 5C and 5D show time-dependent % HiBiT-ACKR4 internalization by ACKR4 antibodies disclosed herein.
  • FIG. 5E shows lytic luminescence of HiBiT-ACKR4 upon treatment with ACKR4 antibody or control after 15 h.
  • FIG. 5F shows % HiBiT-ACKR4 down-regulation by ACKR4 antibody calculated by: 100 X [1 - (RLU of treatment / RLU of Neg Ctl IgG)].
  • Example 10 In vivo Detection of Human Backbone ACKR4 Antibodies in Mouse Serum [00300] Mice were intravenously injected with ACKR4 antibody diluted in 1X PBS at a dose volume of 200 ul per mouse.
  • mice were warmed with a heat lamp, and the tip of the tail was cut with a razorblade and ⁇ 70 ul of blood was collected into an Eppendorf tube.
  • mice were euthanized by CO2 inhalation and ⁇ 300 ul of blood was collected by cardiac puncture using a 25-gauge needle within 5 min of euthanasia.
  • Blood was allowed to clot for 45 min at room temperature and centrifuged for 12 min at room temperature at 6000 RPM in a benchtop centrifuge with a 6- inch rotor.
  • ACKR4 antibody was measured in serum using an anti-human/non-human primate IgG MSD kit [Meso Scale Discovery (MSD)]. Serum was diluted in assay diluent to a concentration that is expected to fall within the standard curve of the assay and then ran 1107368.00126 following the manufacturer’s recommended protocol. Signal was detected using the QuickPlex SQ 120 reader (MSD), and sample quantification was interpolated from the standard curve using MSD Discovery Workbench software (v4.0.12).
  • FIG.6A shows in vivo detection of 20 ug of ACKR4 antibodies in mouse serum of BALB/c mice after seven days.
  • FIG.6B shows in vivo detection of 5 ug of ACKR4 antibody in mouse serum of huACKR4 KI/muACKR4 KO mice (HO) and muACKR4 wild type mice (WT) after seven days.
  • FIG. 6C shows in vivo detection of 250 ug of ACKR4 antibodies in mouse serum of HO mice after seven days.
  • Example 11 In vivo Detection of Inhibition of Chemokine Scavenging by ACKR4 Antibodies as Measured by CCL19 and CCL21 Levels
  • Mice were intravenously injected with ACKR4 antibody diluted in 1X PBS at a dose volume of 200 ul per mouse. Blood was collected at pre-determined time points after injection (2 h-7 days). For mid study blood collection, mice were warmed with a heat lamp and the tip of the tail was cut with a razorblade and ⁇ 70 ul of blood was collected into an Eppendorf tube.
  • mice were euthanized by CO2 inhalation and ⁇ 300 ul of blood was collected by cardiac puncture using a 25-gauge needle within 5 min of euthanasia.
  • Blood was allowed to clot for 45 min at room temperature and centrifuged for 12 min at room temperature at 6000 RPM in a benchtop centrifuge with a 6-inch rotor.
  • the serum supernatant was collected with a pipette, plated in a low volume 96-well PCR plate, and stored at -80°C until the samples were analyzed.

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Abstract

La présente invention concerne des anticorps qui se lient de manière sélective à ACKR4 et des compositions comprenant les anticorps. Les anticorps peuvent inhiber le piégeage de chimiokines ; ou peuvent être capables d'une internalisation ACKR4 ; ou peuvent être capables de réguler à la baisse AKCR4.
PCT/US2024/045377 2023-09-05 2024-09-05 Anticorps anti-ackr4, compositions et utilisations associées Pending WO2025054320A1 (fr)

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