EP4662247A1 - Broadly reactive anti-vhh antibodies - Google Patents

Broadly reactive anti-vhh antibodies

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Publication number
EP4662247A1
EP4662247A1 EP24752603.1A EP24752603A EP4662247A1 EP 4662247 A1 EP4662247 A1 EP 4662247A1 EP 24752603 A EP24752603 A EP 24752603A EP 4662247 A1 EP4662247 A1 EP 4662247A1
Authority
EP
European Patent Office
Prior art keywords
cdr
seq
vhh
antibody
car
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24752603.1A
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German (de)
French (fr)
Inventor
Risini Weeratna
Scott MCCOMB
Anne MARCIL
Joey SHEFF
Mehdi Arbabi-Ghahroudi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Research Council of Canada
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National Research Council of Canada
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Application filed by National Research Council of Canada filed Critical National Research Council of Canada
Publication of EP4662247A1 publication Critical patent/EP4662247A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure describes antibodies that specifically bind to the variable domain of Camelid (e.g. Ilama)-derived single domain antibodies (sdAbA/nH).
  • Camelid e.g. Ilama
  • sdAbA/nH single domain antibodies
  • Chimeric Antigen Receptors are used to redirect immune effector cells, such as T- or NK-cells, to specific targets, such as tumour-associated antigens. They consist of an antigen-binding sequence fused to immune cell activating domains which are expressed in T-, NK or other immune effector cells.
  • the antigen binding sequence can be designed using modified monoclonal antibodies (single-chain Fv, orscFv) ormore recently, single-domain antibodies (sdAb’s or nH’s). In pre-clinical or clinical settings, it is important to detect the level of expression of the CAR transduced/transfected at the cell surface.
  • the cell surface expression of scFv-based CAR molecules can be detected using fluorescently labelled commercially available secondary reagents such as anti-human or anti-mouse IgG (heavy and light) secondary antibodies, which have varying utility depending on the characteristics of the CAR being used.
  • fluorescently labelled commercially available secondary reagents such as anti-human or anti-mouse IgG (heavy and light) secondary antibodies, which have varying utility depending on the characteristics of the CAR being used.
  • IgG heavy and light
  • anti-VnH antibodies mouse anti-camelid single domain antibody antibodies
  • anti-VnH antibodies mouse anti-camelid single domain antibody antibodies
  • additional applications of a high quality anti-sdAb antibody include the detection of Hama-sdAb-based CAR expression on immune cells and ex vivo or in vivo expansion/depletion, targeted depletion of CAR- expressing cells, or re-direction of such sdAb based cellular therapies.
  • such antibodies cross react with the relatively constant framework regions of the VHH protein; this means they have the desirable capacity to bind a wide variety of VHH proteins regardless of variable regions.
  • such antibodies fail to cross react with human antibodies, preventing non-specific binding in cell and tissue staining applications.
  • the anti-VnH antibodies or antigen-binding fragments thereof of the present disclosure may be used, for example, for ex vivo or in vivo detection of a therapeutic comprising llama-derived V H H including but not limited to CAR-T therapeutics.
  • Another application of these antibodies is the detection of camelid VHH proteins bound to cells or tissues for assessment of antigen expression on said cells or tissues or for assessing specificity of antibody binding.
  • these antibodies can be used in certain formats to block such HH CAR activation, or in other formats for activation and expansion of VHH CAR- T consisting of target antigen binding domains consisting of Camelid VHH.
  • the present invention provides a composition comprising an anti-VHH antibody, said anti-VHH antibody comprising an antibody or antigen-binding fragment thereof, that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3), wherein composition comprises: a) a first anti-VHH antibody, said CDR H1 , H2, H3, L1 , L2, and L3 comprising the amino acid sequences: CDR H1 : GYGIS (SEQ ID NO:1), CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2), CDR H3: FYFELAY (SEQ ID NO:3), CDR L1 : RTNLGGNYMY (SEQ ID NO:4), CDR L2: YTSNL
  • an anti-VHH antibody comprising an antibody or antigen-binding fragment thereof, that binds specifically to a Camelid VHH (e.g. single domain antibody (sdAb) domain from Lama glama), wherein the antibody or antigen-binding fragment comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3), wherein said CDR H1 , H2, H3, L1 , L2, and L3 comprise an amino acid sequence comprising:
  • sdAb single domain antibody
  • CDR H2 EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
  • CDR L2 YTSNLAP (SEQ ID NO:5)
  • CDR L3 QQFTSSASTWT (SEQ ID NO:6), respectively; or
  • CDR H2 YISSGGGSTFYPDSVKG (SEQ ID NO:8)
  • CDR H3 QRSDYWFDY (SEQ ID NO:9)
  • CDR L2 NAKTLTE (SEQ ID NO:11), and
  • CDR L3 QHHYGTLFT (SEQ ID NO:12), respectively.
  • the present invention provides a composition comprising two antibodies, the first antibody comprising the following CDR sequences:
  • CDR H2 EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
  • CDR L2 YTSNLAP (SEQ ID NO:5)
  • CDR L3 QQFTSSASTWT (SEQ ID NO:6), respectively; and the second antibody comprising the following CDR sequences:
  • CDR H2 YISSGGGSTFYPDSVKG (SEQ ID NO:8)
  • CDR H3 QRSDYWFDY (SEQ ID NO:9)
  • CDR L2 NAKTLTE (SEQ ID NO:11), and
  • CDR L3 QHHYGTLFT (SEQ ID NO:12), respectively.
  • This composition comprising two antibodies may be particularly advantageous, as it provides broadly reactive antibodies that will react with most or almost all HH molecules.
  • This mixture of antibodies advantageously increases the breadth of reactivity, while preferably maintaining a high- quality defined product.
  • the present invention provides an off-the-shelf screening/detection reagent that is able to detect almost all VHHs, regardless of sequence or specificity. This is thought to be because the framework regions of different VHHs nevertheless have areas with similar or identical sequence identity.
  • Use of two anti-VHHs advantageously provides even broader reactivity than a single anti-VHH antibody.
  • the anti-VHH antibody may further comprise four variable heavy domain framework regions (HFR)(HFR 1 , 2, 3 and 4), wherein said HFR 1 , 2, 3, and 4 comprise an amino acid sequence comprising:
  • HFR 1 QAQLQQSGAELARPGASVRLSCKASGYTFT (SEQ ID NO:13),
  • HFR 2 WVKQRTGQGLEWIG (SEQ ID NO:14),
  • HFR 3 TATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO: 15), and
  • HFR 4 WGQGTLVTVSA (SEQ ID NO:16); or
  • HFR 1 EVQLVESGGGLVQPGGSLKLSCAASGFSFS (SEQ ID NO:17),
  • HFR 2 WFRQTPEKRLEWV (SEQ ID NO:18),
  • HFR 3 GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:19), and
  • HFR 4 WGQGTTLTVSS (SEQ ID NQ:20).
  • the anti-VHH antibody may further comprise four variable light domain framework regions (LFR)(LFR 1 , 2, 3 and 4), wherein said LFR 1 , 2, 3, and 4 comprise an amino acid sequence comprising:
  • LFR 1 EIVLTQFPAIISASLGEKVTMNC (SEQ ID NO:21),
  • LFR 2 WYQQKSDASPRLWIY (SEQ ID NO:22),
  • LFR 3 GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:23), and
  • LFR 4 FGGGTKLEIK (SEQ ID NO:24); or
  • LFR 1 DIQMTQSPASLSASVGETVTITC (SEQ ID NO:25),
  • LFR 2 WFQQRQGKSPHLLVY (SEQ ID NO:26),
  • LFR 3 GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC (SEQ ID NO:27), and
  • LFR 4 FGSGTKLEIK (SEQ ID NO:28).
  • the present invention provides an anti-VHH antibody, consisting of a light chain only antigen-binding antibody fragment that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises an amino acid sequence comprising three variable light domain CDR (CDR L1 , L2 and L3), consisting of either:
  • CDR L3 QQFTSSASTWT (SEQ ID N0:6), respectively; or
  • CDR L2 NAKTLTE (SEQ ID NO:11), and
  • CDR L3 QHHYGTLFT (SEQ ID NO:12), respectively.
  • the present invention provides an anti-VHH antibody, consisting of a heavy chain only antigen-binding antibody fragment that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises an amino acid sequence comprising three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), consisting of either:
  • CDR H2 EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
  • CDR H3 FYFELAY (SEQ ID NO:3), respectively;
  • CDR H2 YISSGGGSTFYPDSVKG (SEQ ID NO:8)
  • CDR H3 QRSDYWFDY (SEQ ID NO:9), respectively.
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising:
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30).
  • VH variable heavy domain
  • the anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising:
  • the anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
  • the anti-VHH antibody may comprise a variable heavy domain ( H) comprising amino acid sequence comprising:
  • variable light domain comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30), and a variable light domain (VL) comprising amino acid sequence comprising:
  • DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
  • the antibody or antigen-binding fragment may be an IgA, IgD, IgE, IgG, or IgM.
  • the antigen-binding fragment may be a single-domain antibody (sdAb), or a singlechain variable fragment (scFv).
  • sdAb single-domain antibody
  • scFv singlechain variable fragment
  • the sdAb may comprise three CDR (CDR1 , 2 and 3) comprising SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, respectively, or comprising SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.
  • the antibody or an antigen-binding fragment thereof may be humanized or partially humanized.
  • the antibody or antigen-binding fragment may be operable to stimulate or block activation, expansion, or both activation and expansion of an sdAb-based chimeric antigen receptor (CAR)-T cell.
  • CAR sdAb-based chimeric antigen receptor
  • the antibody or antigen-binding fragment is bound directly or through a linker to a functional moiety.
  • the antibody or antigen-binding fragment may be linked to the functional moiety via a peptide linker.
  • the peptide linker may comprise 1-40 amino acid residues.
  • the peptide linker may comprise the amino acid sequence (SS) n , (GGG) n , (GGGG) n , (GGGS) n , or (SSGGG) n , (SEQ ID NO: 33), or (GGGGS) n (SEQ ID NO: 34), wherein n > 1 .
  • the functional moiety may be a detectable label, a peptide, a polypeptide, a protein, an enzyme, a nucleic acid, a lipid particle, a cytotoxic agent, a drug, a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof; wherein the antibody or antigen-binding fragment is functional and wherein the linked detectable label, peptide, polypeptide, protein, enzyme, nucleic acid, lipid particle, cytotoxic agent, drug, second antibody, antibody fragment, second antigen-binding fragment, or the combination of any two or more thereof is functional.
  • the detectable label may be a fluorescent marker, a radioactive marker, an MRI contrast agent, a detectable secondary antibody, or combinations thereof.
  • composition comprising the anti- VHH antibody of the present invention, and a pharmaceutically acceptable diluent, carrier or excipient.
  • nucleic acid molecule encoding an anti-VHH antibody of the present invention.
  • a vector comprising the nucleic acid molecule of the present invention operably linked to one or more regulatory elements to allow expression of the antibody or antigen-binding fragment of the present invention in a host cell.
  • a cell comprising the vector of the present invention for expressing the anti-VHH antibody of the present invention.
  • a method for the detection of the surface presence of a sdAb domain from Lama glama on a cell comprising detecting an anti-VHH antibody of the present invention, or the compound of the present invention contacting said cell.
  • the anti-VHH antibody of the present invention, or the compound of the present invention may comprise a detectable label such as Alexa FluorTM 647 shown in examples herewith.
  • the cell may be a chimeric antigen receptor (CAR)-T cell.
  • CAR chimeric antigen receptor
  • a method for the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell comprising a CAR comprising a sdAb domain from Lama glama, comprising contacting said CAR-T cell with an anti-VHH antibody of the present invention.
  • CAR chimeric antigen receptor
  • a method for redirection of a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR comprising a sdAb domain from Lama glama comprising contacting said CAR-T cell with the compound of the present invention, the compound comprising a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof, specific for a second antigen different from the antigen for which the CAR is specific.
  • the present invention provides an anti-VHH antibody operable
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • the present invention provides a method for the detection of a VHH on the surface a cell comprising detecting said VHH using the anti-VHH antibody described herein.
  • the cell is a CAR-T cell.
  • the anti-VHH antibody comprises a detectable label.
  • the present invention provides a method for either stimulating or blocking the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell having a VHH on the surface, comprising contacting said CAR-T cell with the the composition or anti-VHH antibody as described herein.
  • CAR chimeric antigen receptor
  • the present invention provides a method for redirection of a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR with a surface VHH, comprising contacting said CAR-T cell with the composition or anti-VHH antibody described herein, wherein the anti-VHH antibody comprises a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof, specific for a second antigen different from the antigen for which the CAR is specific.
  • CAR chimeric antigen receptor
  • the present invention provides a method for assessing the binding properties of a VHH, comprising subjecting a sample in vivo, ex vivo, or in vitro to purified VHH and then detecting binding of said VHH using the composition or anti-VHH antibody described herein.
  • the present invention provides a use of the anti-VHH antibody described herein to directly or indirectly detect VHH proteins as a method of assessing the binding properties of VHH proteins.
  • the present invention provides an anti-VHH antibody or composition as described herein, wherein the anti-VHH antibody is operable to stimulate or block activation, expansion, or both activation and expansion of an sdAb-based chimeric antigen receptor (CAR)-T cell.
  • CAR chimeric antigen receptor
  • Fig. 1 illustrates binding of eight hybridoma supernatants against a panel of sdAb- based CARs Generated using 2 separate human donor T cells. Un-transduced T cells and two scFv- based CARs (CD22-m971 and CD19-FMC63) were used as controls.
  • Fig. 2 illustrates CAR expression detected by evaluating GFP expression versus binding of the anti-VnH mAb 2A3. Un-transduced T cells (Mock), CD19-scFv, and BCMA-scFv based CAR were used as negative controls, where no binding was expected.
  • Fig. 3 illustrates amino acid sequences of H and VL and CDR regions of anti-VnH antibodies 2A3 and 3H12.
  • FIG. 4 illustrates binding of hybridoma expressed versus recombinantly expressed mouse lgG2a formats of the anti-V H H monoclonal antibodies to three different llama-V H H proteins.
  • Fig. 5A illustrates detection of sdAb-based CAR expression on Jurkat T cells using unlabelled recombinant anti-V H H antibody 2A3, as well as the long-term stability of the anti-V H H antibody when stored at -80°C.
  • Fig. 5B illustrates detection of sdAb-based CAR expression on Jurkat T cells using unlabelled recombinant anti-VnH antibody 3H12 as well as the long-term stability of the anti-VnH antibodies when stored at -80°C.
  • Fig. 6 illustrates the results of hydrogen-deuterium exchange mass spectrometry experiment, which identifies the specific interaction peptides for the anti-VHH antibodies. This experiment reveals two partially overlapping, but distinct for the 2A3 and 3H12 antibodies.
  • Fig 7. Illustrates the strength of binding for the 2A3 and 3H12 anti-VHH antibodies individually or in a 1 :1 cocktail against 76 unique VHH proteins, as assessed via ELISA. These results reveal negative binding of an antibody mixture for only 1 of 76 VHH proteins, with the mixture consistently outperforming the binding of either antibody alone.
  • Fig. 8 illustrates the comparability of two different batches of Alexa FluorTM (AF)-467 labelled anti-V H H antibodies for their ability to detect expression of sdAb-based CAR constructs on T cells.
  • AF Alexa FluorTM
  • This figure also demonstrates that a mixture of two anti-VnH antibodies (3H12 and 2A3) can be effectively employed to stain cells with surface expression of sdAb-CARs.
  • FIG. 9 illustrates comparability of sdAb-based CAR detection using AF-647-conjugated F233-2A3-2 (left panel) or FITC-conjugated CD22 protein (ACRO BioSystems) (right panel).
  • healthy donor enriched CD4/CD8 lymphocytes that were 1-day post-activation with TransAct (10 pl/10 6 cells) were transduced with sdAb-based CAR.
  • Transduction was performed in 4x24-well plates (at 1x10 6 cells/well) then expanded in Grex flasks to ⁇ 650 x10 6 cells at day 14. Cells were then stained with AF-647-conjugated F233-2A3-2 or FITC-conjugated CD22 protein (ACRO).
  • Fig. 10 illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of CD22-specific 1 ug13 and 1 ug36 to CD22-expressing Ramos cells but not CD22-knockout Ramos cells is shown. A negative control sdAb specific for an irrelevant bacterial protein (B131) was also used to demonstrate the specificity of sdAb binding.
  • Fig. 11 further illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of BCMA- specific sdAbs to BCMA-high RPMI8226 cells, BCMA-low Raji cells, or BCMA-negative Jurkat cells is shown.
  • Fig. 12 further illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of mesothelin- specific sdAbs to mesothelin-high H292 cells is shown.
  • Fig 13. illustrates the application of anti-VnH antibodies as secondary labelling reagent in tissue and cell level binding in an immunohistochemical assessment of sdAb specificity. Specifically, a frozen tissue microarray was stained with either CD22-specific sdAb or an irrelevant specificity control sdAb, followed by staining with secondary anti-V H H, and eventually a tertiary anti-mouse detection reagent. Staining results for representative tissues are shown here.
  • Fig. 14A illustrates the activation of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells by anti-CD3 antibody OKT3.
  • Fig. 14B illustrates the activation of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells by anti-VnH monoclonal antibody 3H12.
  • Fig. 15 illustrates an expansion of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells following simulation with surface immobilized anti-VnH monoclonal antibody 3H12. All anti-VnH stimulated sdCAR cells expanded for at least 15 days, with only transient expansion on scFvCAR or untransduced T cells due to previous polyclonal activation.
  • Fig 16 illustrates a diagram of the potential mode by which a soluble anti-VHH antibody might interfere with and inhibit CAR activation.
  • Fig 17. Illustrates the inhibition of CAR reactivity in the presence of various doses of 2A3 or 3H12 anti-VHH antibodies, or a combination thereof. Specifically, Jurkat cells engineered to express an EGFR-directed CAR were combined with EGFR-expressing CAR cells, with varying doses of the anti-VHH antibodies, a mixture of the antibodies, or vehicle control.
  • Fig. 18 illustrates the loss in viability observed following treatment of VHH-CAR expressing Jurkat cells but not WT Jurkat cells following treatment with anti-VHH antibody drug conjugate.
  • Fig. 19 illustrates a molecular strategy wherein the anti-VHH molecule is used to redirect the CAR towards additional target molecules.
  • the present invention is directed to a technology for the specific recognition of the variable domain of a single domain antibody (sdAb) from Lama glama.
  • sdAb single domain antibody
  • VHH single domain antibody
  • the anti-VHH antibody or antigen-binding fragment comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3).
  • CDR variable heavy domain complementarity determining regions
  • the CDR H1 , H2, H3, L1 , L2, and L3 of the invention may comprise the following amino acid sequence comprising: 1) CDR H1 : GYGIS (SEQ ID N0:1), CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID N0:2), and CDR H3: FYFELAY (SEQ ID N0:3); and CDR L1 : RTNLGGNYMY (SEQ ID N0:4), CDR L2: YTSNLAP (SEQ ID N0:5), and CDR L3: QQFTSSASTWT (SEQ ID N0:6), respectively; or or
  • CDR H1 NYHMS (SEQ ID NO:7), CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8), and CDR H3: QRSDYWFDY (SEQ ID NO:9); and CDR L1 : RASENIYSYLA (SEQ ID NQ:10), CDR L2: NAKTLTE (SEQ ID NO:11), and CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively.
  • the anti-VHH antibody may further comprising fourvariable heavy domain framework regions (HFR)(HFR 1 , 2, 3 and 4).
  • HFR fourvariable heavy domain framework regions
  • the HFR 1 , 2, 3, and 4 may comprise the following amino acid sequence comprising:
  • HFR 1 QAQLQQSGAELARPGASVRLSCKASGYTFT (SEQ ID NO:13), HFR 2: WVKQRTGQGLEWIG (SEQ ID NO:14), HFR 3: TATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO:15), and HFR 4: WGQGTLVTVSA (SEQ ID NO: 16).
  • HFR 1 EVQLVESGGGLVQPGGSLKLSCAASGFSFS (SEQ ID NO:17), HFR 2:
  • GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:19), and HFR 4: WGQGTTLTVSS (SEQ ID NQ:20).
  • the anti-VHH antibody may further comprising fourvariable light domain framework regions (LFR)(LFR 1 , 2, 3 and 4).
  • LFR 1 , 2, 3, and 4 may comprise the following amino acid sequence comprising:
  • LFR 1 EIVLTQFPAIISASLGEKVTMNC (SEQ ID NO:21), LFR 2: WYQQKSDASPRLWIY (SEQ ID NO:22), LFR 3: GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:23), and LFR 4: FGGGTKLEIK (SEQ ID NO:24); or
  • LFR 1 DIQMTQSPASLSASVGETVTITC (SEQ ID NO:25)
  • LFR 2 WFQQRQGKSPHLLVY (SEQ ID NO:26)
  • LFR 3 GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC (SEQ ID NO:27)
  • LFR 4 FGSGTKLEIK (SEQ ID NO:28).
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29).
  • VH variable heavy domain
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NO:30).
  • VH variable heavy domain
  • the anti-VHH antibody may comprise a variable light domain (V ) comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
  • the anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
  • VL variable light domain
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29), and a variable light domain (V ) comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
  • VH variable heavy domain
  • VH variable heavy domain comprising amino acid sequence comprising: QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYY
  • the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30), and a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
  • VH variable heavy domain
  • VL variable light domain
  • the anti-VHH antibody may be single-domain antibody (sdAb), an engineered single-domain fragment, a fragment antigen-binding (Fab), a single-chain variable fragment (scFv), or a single-chain fragment antigen-binding (scFab).
  • the antibody or antigen-binding fragment may be an IgA, an IgD, an IgE, an IgG, or an IgM.
  • the sdAb may comprises three CDR (CDR1 , 2 and 3) comprising SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, respectively, or comprising SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, that binds specifically to a single domain antibody (sdAb) domain from Lama glama.
  • CDR1 , 2 and 3 comprising SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, respectively, or comprising SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, that binds specifically to a single domain antibody (sdAb) domain from Lama glama.
  • the expression “substantially identical sequence” is intended to mean an amino acid sequence which may comprise one or more conservative amino acid mutations.
  • a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).
  • one or more conservative amino acid mutations may be made to the one or more framework regions of the anti-VHH antibody while maintaining both the CDR sequences and the overall structure of the CDR of the antibody or antigen-binding fragment; thus the specificity and binding of the antibody or antigen-binding fragment are maintained.
  • one or more conservative amino acid mutations may be made to the one or more framework regions of the anti-VHH antibody and to a CDR sequence while maintaining the antigen-binding function of the overall structure of the CDR of the antibody or antigen-binding fragment; thus the specificity and binding of the antibody or antigen-binding fragment are maintained.
  • a conservative mutation may be a conservative amino acid substitution.
  • Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another amino acid of the same group.
  • basic amino acid it is meant a hydrophilic amino acid having a side chain pK value of greater than 7, which is typically positively charged at physiological pH.
  • Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K).
  • neutral amino acid also “polar amino acid”
  • polar amino acid it is meant a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q).
  • hydrophobic amino acid (also “non-polar amino acid”) it is meant an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984).
  • Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Vai or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).
  • “Acidic amino acid” refers to a hydrophilic amino acid having a side chain pK value of less than 7, which is typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E) and aspartate (Asp or D).
  • Sequence identity is used to evaluate the similarity of two sequences. It is determined by calculating the percentage of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.
  • the substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or any percentage there between, at the amino acid level to sequences described herein.
  • a substantially identical sequence retains the activity and specificity of the reference sequence.
  • the difference in sequence identity may be due to one or more conservative amino acid mutations.
  • the present invention may be directed to an antibody or antigen-binding fragment comprising a sequence at least at least 95%, at least 98%, or at least 99% identical to that of one or more of the antibodies or antigen-binding fragments described herein.
  • immunoglobulin refers to an antigen-binding protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM.
  • VL variable
  • CL constant
  • CH1 , CH2, CH3 constant
  • Fv antigen binding region
  • the light and heavy chain variable regions are responsible for binding a target antigen and can therefore show significant sequence diversity between antibodies.
  • the constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.
  • the variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen.
  • the majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (VH) and light (VL) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant.
  • the specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen.
  • Various schemes exist for identification of the regions of hypervariability the two most common being those of Kabat and of Chothia and Lesk.
  • Kabat and Wu (1991) define the “complementarity-determining regions” (CDRs) based on sequence variability at the antigen-binding regions of the VH and VL domains.
  • CDRs complementarity-determining regions
  • Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the VH and L domains.
  • CDR and “hypervariable loop” interchangeably, and they may be so used herein.
  • the CDRs/loops are identified herein according to the IMGT nomenclature scheme (i.e., CDR1 , 2 and 3, for each variable region).
  • an “antibody fragment” or “antigen-binding fragment” as referred to herein may include any suitable antigen-binding antibody fragment known in the art.
  • the antibody fragment may be a naturally-occurring antibody fragment, or it may be a non-naturally occurring antibody fragment obtained, for example, by manipulation of a naturally-occurring antibody or by recombinant methods.
  • an antibody fragment may include, but is not limited to, a Fv, a single-chain Fv (scFv; a molecule consisting of VL and VH connected with a peptide linker), a Fab, a F(ab’)2, single-domain antibody (sdAb; a fragment composed of a single VL or VH or a VHH), or a multivalent presentation of any of these.
  • Antibody fragments such as those just described may require one or more linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches for their construction.
  • anti-VHH antibody comprises both “antibodies” (such as monoclonal antibodies) as well as “antigen-binding fragments”, unless specified otherwise.
  • the antigen-binding fragment of the present invention may be a sdAb derived from a naturally-occurring source.
  • Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed VHH.
  • SdAbs have also been observed in shark and are termed VNAR (Nuttall et al, 2003).
  • Other sdAbs may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005).
  • sdAb includes an sdAb directly isolated from a VH, VHH, VL, OTVNAR reservoir of any origin through phage display or other technology, an sdAb derived from the aforementioned sdAb, a recombinantly produced sdAb, as well as an sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.
  • SdAbsA/nHs possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997). They can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of one or more non-canonical disulfide bonds (Hussack et al, 2011a, b; Kim et al, 2012), may also be brought to the sdAb.
  • An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site.
  • not all CDRs may be required for binding the antigen.
  • one, two, or three of the CDRs may contribute to binding and recognition of the antigen by the sdAb of the present invention.
  • the CDRs of the sdAb, VHHS or variable domain are referred to herein as CDR1 , CDR2, and CDR3.
  • the present invention further encompasses an antibody or an antigen-binding fragment that is “humanized” using any suitable method known in the art, such as, but not limited to, CDR grafting or veneering.
  • Humanization of an antibody or an antigen-binding fragment comprises replacing an amino acid in the antibody or antigen-binding fragment sequence with its human counterpart, as found in the human consensus sequence, without substantial loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or antigen-binding fragment when introduced into a human subject.
  • one or more than one of the CDRs defined herein may be fused or grafted to a human variable region (VH, orVi.), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to a human antibody fragment framework region (Fv, scFv, Fab) or to another protein of similar size and nature onto which a CDR can be grafted (Nicaise et al, 2004).
  • VH, orVi. human variable region
  • IgA, IgD, IgE, IgG, and IgM human antibody fragment framework region
  • Fv, scFv, Fab human antibody fragment framework region
  • TGF-p family member a human/rhesus/rat/mouse TGF-p family member, collectively referred to as TGF-p family member
  • CDR grafting is known in the art and is described in at least the following: US Patent No. 6180370, US Patent No. 5693761 , US Patent No. 6054297, US Patent No. 5859205, and European Patent No. 626390.
  • Veneering also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of an antibody or antigen-binding fragment; thus, preserving buried nonhumanized residues, which may be important for CDR conformation, while minimizing the potential for immunological reaction against solvent-exposed regions. Veneering is known in the art and is described in at least the following: US Patent No. 5869619, US Patent No. 5766886, US Patent No. 5821123, and European Patent No. 519596. Persons of skill in the art would also be amply familiar with methods of preparing such humanized antibody fragments and humanizing amino acid positions. [0094] According to an embodiment, the antibody or antigen-binding fragment of the present invention may be operable to stimulate activation, expansion, or both activation and expansion of an sdAb chimeric antigen receptor (CAR)-T cell.
  • CAR sdAb chimeric antigen receptor
  • the antibody or antigen-binding fragment according to the present invention may comprise one or more additional sequences to aid in expression, detection or purification of the antibody or antigen-binding fragment.
  • Any such sequence or tag known to those of skill in the art may be used.
  • the antibody or antigen-binding fragment may comprise a targeting or signal sequence (such as, but not limited to, ompA or pelB), a detection/purification tag (such as, but not limited to, c-Myc, HA, His5, or His6), or a combination of any two or more thereof.
  • the additional sequence may be a biotin recognition site such as that described by Cronan et al.
  • a linker sequence may be used in conjunction with the additional sequence or tag, or may serve as a detection/purification tag.
  • linker sequence is intended to mean a short (typically 40 amino acids or fewer) peptide sequence that is introduced between protein domains. Linker sequences are often composed of flexible residues such as glycine and serine so that the linked protein domains are free to move relative to one another.
  • the linker sequence can be any linker sequence known in the art that would allow for the antibody and the functional moiety of the present invention to be operably linked for the desired function.
  • the linker may be any sequence known in the art (either a natural or synthetic linker) that allows for an operable fusion comprising an antibody or antigen-binding fragment linked to a polypeptide (e.g., the functional moiety).
  • the linker sequence may be a linker sequence L such as (SS) n , (GGG) n , (GGGG) n , (GGGS) n , (SSGGG) n , (SEQ ID NO: 33), or (GGGGS)n (SEQ ID NO: 34) wherein n is equal to or greater than 1 , or from about 1 to about 5, or from about 1 to 15; or n may be any number that would allow for the operability of the compound of the present invention.
  • the linker may be an amino acid sequence, for example, an amino acid sequence that comprises about 1 to about 40 amino acids, or about 3 to about 40 amino acids, or about 5 to about 40 amino acids, or about 10 to about 40 amino acids, or about 15 to about 40 amino acids, or about 20 to about 40 amino acids, or about 25 to about 40 amino acids, or about 30 to about 40 amino acids, or about 35 to about 40 amino acids, or about 3 to about 35 amino acids, or about 5 to about 35 amino acids, or about 10 to about 35 amino acids, or about 15 to about 35 amino acids, or about 20 to about 35 amino acids, or about 25 to about 35 amino acids, or about 30 to about 35 amino acids, or about 3 to about 30 amino acids, or about 5 to about 30 amino acids, or about 10 to about 30 amino acids, or about 15 to about 30 amino acids, or about 20 to about 30 amino acids, or about 25 to about 30 amino acids, or about 3 to about 25 amino acids, or about 5 to about 25 amino acids, or about 10 to about 25 amino acids, or about 15 to about 25 amino acids, or about
  • the antibody or antigen-binding fragment of the compound may be covalently bound directly or through a linker sequence to a detectable label (e.g., a fluorescent marker, for example Alexa FluorTM 647), a radioactive marker, an MRI contrast agent, a detectable secondary antibody, or combinations thereof, to co-localize the therapeutic agent of the present invention to an anatomical location where the sdAb-CAR modified immune cells would home to, and for detection of sdAb-CAR modified immune cells), a peptide, a polypeptide (e.g.
  • growth factor CIBP2 an antimicrobial cyclic peptide
  • a protein an enzyme [such as iduronate-2-sulfatase (IDS), acid beta-glucosidase (GCase), a serine protease, a growth factor, etc.], another (or the same) antibody or a fragment operable to bind a target epitope (e.g.
  • IDS iduronate-2-sulfatase
  • GCase acid beta-glucosidase
  • serine protease a growth factor, etc.
  • another (or the same) antibody or a fragment operable to bind a target epitope e.g.
  • an anti-microbial antibody an anti-inflammatory antibody, an intrabody, a BBB-crossing antibody, a neurodegeneration target antibody, an ion channel targeting antibody, a cancer associated antigen antibody, a checkpoint inhibitor targeting antibody, or a GPCR targeting antibody
  • a nucleic acid e.g., an siRNA, an mRNAs, an aptamer, etc.
  • a lipid particle e.g., a lipid micro- or nano- particle loaded with a given payload, such as a peptide, a polypeptide, a compound, a nucleic acid, etc.
  • a cytotoxic agent i.e ., for elimination of sdAb-based CAR modified immune cells in the event of undesirable clinical outcome
  • a combination of any two or more thereof in which both the antibody or antigen-binding fragment and the rest of the compound (i.e., the functional moiety) remain functional for their intended
  • the compound may be fused or linked to a second antibody or antigen-binding fragment, operable to bind a target epitope, which may be the same as, or distinct from the epitope of the antibody or antigen-binding fragment of the present invention.
  • the antibody or antigen-binding fragment of the present invention may also be in a multivalent display format, also referred to herein as multivalent presentation. Multimerization may be achieved by any suitable method known in the art.
  • a multivalent display format may encompass a chimeric or humanized format of VHH of the present invention linked to an Fc domain, or bi or tri-specific antibody fusions with two or three VHHS recognizing unique epitopes.
  • Such antibodies are easy to engineer and produce, can greatly extend the serum half-life of a sdAb, and may be excellent tumor imaging reagents (Bell et al., 2010).
  • the Fc domain in the multimeric complex as just described may be any suitable Fc fragment known in the art.
  • the Fc fragment may be from any suitable source; for example, the Fc fragment may be of mouse or human origin.
  • the Fc fragment may be a mouse Fc2b fragment or a human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010).
  • the Fc fragment may be fused to the N-terminal or C-terminal end of the VHH or humanized version of the present invention.
  • Each subunit of the multimers described above may comprise the same or different antibodies or antigen-binding fragments of the present invention, which may have the same ordifferent specificity. Additionally, the multimerization domains may be linked to the antibody or antigen-binding fragment using a linker, as required; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules but should not hamper the antigenbinding properties of the antibody or antigen-binding fragment.
  • the linker sequence can be any linker known in the art that would allow for the compound of the present invention to be prepared and be operable for the desired function.
  • the present invention also encompasses a composition comprising one or more than one anti-VHH antibody of the present invention and/or compound of the present invention as described herein.
  • the composition may comprise a single anti- VHH antibody and/or compound as described above, or the composition may comprise a mixture of anti-VHH antibody and/or compounds.
  • the anti-VHH antibody and/or compounds may have the same specificity, or they may differ in their specificities; for example, and without wishing to be limiting in any manner, the composition may comprise anti-VHH antibody and/or compounds specific to a single domain antibody domain of Lama glama (same or different epitope).
  • the carrier when the composition is provided in suspension form, may comprise water, saline, or a suitable buffer, and optionally comprise one or more additives to improve solubility and/or stability. Reconstitution to produce a suspension may be effected in a buffer at a suitable pH to ensure the viability of the antibody orantigen- binding fragment. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be formulated for delivery of the antibody or antigen-binding fragment to the gastrointestinal tract of the subject.
  • composition may comprise encapsulation, time release, or other suitable technologies for delivery of the anti-VHH antibody and/or compound of the present invention. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present sdAb and/or compound.
  • a method for the detection of the surface expression of a sdAb domain from Lama glama on a cell comprising detecting the anti-VHH antibody of the present invention, the compound of the present invention, contacting the cell.
  • the anti- VHH antibody, or the compound may comprises a detectable label which is Alexa FluorTM 467.
  • the cell may be a chimeric antigen receptor (CAR)-T cell.
  • an anti- VHH antibody or a mixture of anti-VHH antibodies which have been chemically conjugated to a desired payload molecule for targeted delivery to a cell which expresses a chimeric antigen receptor incorporating a sdAb domain from Lama glama as the antigen-binding fragment thereof.
  • the payload molecule could be, but is not limited to, molecules that are chemicals, radiochemicals, nucleotides, nucleic acids, lipid nanoparticles (LNP),, peptides, or proteins, intended to achieve a desired biological effect within targeted VHH-CAR expressing cells. This would include drugs, like radiopharmaceuticals and cytotoxic drugs.
  • the payload could be a cytotoxic molecule.
  • mice Four six-week old female A/J mice (The Jackson Laboratory, Bar Harbor, ME) were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 pg of sdAb FC5- MOD antigen emulsified in Titermax adjuvant (Cedarlane Labs, Burlington, ON) at day 0 and in PBS without adjuvant at day 26. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) at day 33, and serum was stored at -20°C until further use.
  • Titermax adjuvant Cedarlane Labs, Burlington, ON
  • microplates were washed 4 times with PBS-Tween 200.05% and 25 pl of a 1/5.000 dilution of alkaline phosphatase conjugated goat anti-mouse IgG (H+L) (#115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added.
  • microplates were washed 4 times and 25 pl of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for 30 min.
  • pNPP p-nitrophenyl phosphate
  • HAT selection medium IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat#P7539), 1 ng/ml mouse IL-6 (Biolegend Cat#575706), HAT media supplement (Sigma Cat#H0262) and L-glutamine (Hy-Clone Cat#SH30034.01) and incubated at 37°C, 5% CO2.
  • HAT selection medium IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat#P7539), 1 ng/ml mouse IL-6 (Biolegend Cat#575706), HAT media supplement (Sigma Cat#H0262) and L-glutamine (Hy-Clone Cat#SH30034.01)
  • Hybridoma supernatants were screened by ELISA to detect specific binders.
  • 96-well half-area plates (Costar #3690) were coated with 25 pl of FC5-MOD or FC5 derivatives FC5-V H H or FC5-hFc-1X0, or P257 (unrelated sdAb) or EG2-hFc-X2 (unrelated sdAb) or BSA (negative control) at 5 pg/ml in PBS and incubated overnight at 4°C.
  • Microplates were washed 3 times with PBS, blocked with PBS-BSA 1%, and 25 pl of hybridoma supernatants were added and incubated at 37°C, 5% CO2 for 2 hours.
  • Hybridoma supernatants were further analyzed by ELISA for positive reactivity to various sdAb proteins: FC5-MOD (immunogen), sdAb-1 ug13, and sdAb-1 ug36; and for negative reactivity to human antibodies using protX-hlgG (negative control). All 8 mAbs were found positive for sdAb-1 ug13 and -1 ug36 as well as for the original immunogen (positive control) and negative for protX- hlgG antigen (Table 2).
  • a panel of eight hybridoma supernatants were selected based on Jurkat-CAR binding results to assess their ability to bind human primary T cell derived sdAb-based CARs by flow cytometry. Hybridoma supernatants were normalized by antibody concentration. Primary CAR-T cells generated using T cells from 2 separate donors were incubated with the hybridoma supernatants, washed, and incubated with AF-647 anti-mAb Fab 2° antibody. Fig. 1 shows the binding of the eight hybridoma across 5 different sdAb-based CAR; all targeting human CD22. Untransduced T cells and two scFv- based CARs (CD22-m971 and CD19-FMC63) were used as controls.
  • Antigens at 0.3-1.2 mg/ml were incubated at 95°C for 5 min in PBS containing DTT at a final concentration of 40 mM. They were then incubated on ice for 5 min and diluted at their final coating concentration (5 pg/ml) for ELISA purpose.
  • Selected anti-sdAb mAbs F233-2A3 and F233-3H12 were further characterized by ELISA on human immunoglobulins to assess their specificity to sdAbs only.
  • ELISA was performed as described above with immobilized human lgG1 , lgG2, lgG3, lgG4, IgA, slgA and IgM along with positive control sdAb-1 ug13 and -1 ug36.
  • 3E6 is an anti-GFP negative control, and anti-hlgG (H+L) was used as a positive control for all human Ig.
  • This assay was conducted using one set concentration (30nM) of 2A3. Increasing the 2A3 antibody concentration may be effective for increasing the binding to the weaker HH.
  • V H and V of each mAbs were sequenced by Sanger sequencing. Briefly, mRNA was extracted from hybridoma clones (Dynabeads mRNA Direct kit, ThermoFisher Scientific) and reverse transcribed into cDNA (Maxima H Minus First Strand cDNA with dsDNAse, ThermoFisher Scientific®). DNA encoding H and VL domains was PCR amplified (Q5 Hot Start High-Fidelity DNA PolymeraseTM, NEBTM) using mixtures of degenerate forward primers annealing in FR1 and a single reverse primer annealing in CH1 (NovagenTM Mouse Ig Heavy, Kappa and Lambda Primer sets).
  • the resulting amplicons were sequenced by regular Sanger sequencing using their respective forward and reverse primers.
  • the DNA sequence of each H and VL domains were analyzed and then translated in silica.
  • the CDRs sequence were determined using Kabat CDR numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi).
  • the amino-acid sequences of the VH and VL are shown in Fig. 3.
  • the sequence analysis revealed that clones 2A3 and 3H12 have unique VH and VL CDRs.
  • the anti-sdAbs monoclonal antibodies identified in Example 2 were produced recombinantly in CHO C3117 or CHO 55E1 cells as stable pools.
  • V H and V regions were cloned as fusions with mouse lgG2a/kappa constant regions (mouse lgG2a heavy chain and mouse kappa light chain, respectively) into the pTT109 vector.
  • the amino acid sequences of the recombinant antibodies recF233-2A3-mlgG2a and recF233-3H12-mlgG2a is provided in the sequence table (SEQ ID NOs: 36, 37, 38 and 39, respectively). All light chain sequences comprise a signal sequence MRLPAQLLGLLMLWVSGSSG (SEQ ID NO:40) at the N-terminus, while heavy chain sequences comprised the signal sequence MPLLLLLPLLWAGALA (SEQ ID NO:41) at the N-terminus.
  • Recombinant antibody expression was validated via a 2 mL expression scout. Briefly, CHO cells were transfected with VL and VH containing constructs (1 :1 ratio).
  • CM Conditioned medium
  • Table 5 Apparent KD of anti-sdAb mAbs as determined by ELISA analysis.
  • the recombinant anti-VnH mAb was also tested for their ability to detect surface expression of sdAb-based CAR constructs as well as for their stability when stored over time at -80°C. To perform this assessment, serial dilution of the recombinant antibodies was added to 96-well plate followed by addition of respective CAR-T cells. The cells were allowed to incubate with the antibody for 15 minutes at room temperature. Cells were then centrifuged, washed and the secondary detection antibody; Goat F(ab') 2 anti-mouse IgG (H+L) Alexa-FluorTM 647 was added to the cells.
  • Fig. 5 depicts the binding of the anti-VnH mAb 2A3 and 3H12 to Jurkat T cells stably expressing sdCD22 CAR constructs. The binding curves for each antibody at time of production and at various time points post storage at -80°C are depicted. Results demonstrate strong and dose dependent binding of both 2A3 and 3H12 anti-V H H mAb to CD22 sdAb based CAR constructs expressed on Jurkat T cells. Results demonstrate that these antibodies show similar binding kinetics and thus are stable for at least 12 months when stored at -80°C.
  • ANTI-VHH ANTIBODIES SHOW DIVERGENT EPITOPE BINDING PROPERTIES AS ASSESSED VIA HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY
  • Recombinant anti-VHH antibody samples were produced as described in example 6 and utilized here.
  • Purified sdAb-1 ug36 protein was equilibrated with a saturating concentration of each anti-VHH antibody at a 1 :1 molar ratio.
  • Deuterium labelling of the antibody-VHH complexes or sdAb- 1 ug36 alone was initiated by a two-fold dilution with deuterated buffer (phosphate buffered saline in 90% D2O). A controlled time period of either 0.5 minutes or 3 minutes was allowed before the reaction was quenched by a 5-fold dilution with 1 M Guanidine HCI 40mM TCEP in 0.1% formic acid at 4°C.
  • Fig. 6 depicts the difference in the relative level of deuterium between the sdAb-1 ug36/anti- VHH complexes and unbound sdAb-1 ug36 observed via a mass shift in specific peptides within the analyzed sdAb-1 ug36 sequence.
  • ANTI-VHH ANTIBODIES SHOW BROAD REACTIVITY WITH DIVERSE LLAMA VHH PROTEINS VIA ELISA
  • VHH protein was passively absorbed on a 96-well Immulon 4HBX plate in PBS buffer at 4°C overnight before blocking with PBS + 5% milk.
  • Anti-VHH antibodies were then added to the plate at a known concentration and incubated at room temperature for 60 mins to allow antibody-antigen interaction. Plates were then washed 3 times with PBS + 0.05% Tween20 buffer. The amount of bound anti-VHH antibody was detected using anti-mouse HRP antibody, followed by washing. Finally, the TMB buffer was added for quantitation of HRP bound to the plate, the reaction was stopped with 1 M sodium phosphate, and assessed using a plate reader.
  • Fig. 7 depicts the amount of bound anti-VHH antibody detected for 2A3, 3H12, or a cocktail of both antibodies against 76 different VHH proteins.
  • Results show shows 75 of 76 unique VHH’s are recognized by a cocktail 1 :1 mix of the anti-VHH antibodies; 74 of 76 are recognized by 3H12 mAb; and 73 of 76 are recognized by 2A3 mAb.
  • Results demonstrate that 2A3 and 3H12 both show broad reactivity to many llama VHH proteins, with distinct binding patterns.
  • a 1 :1 mix of both antibodies provides the best overall breadth of binding to diverse VHH proteins.
  • Antibody samples (2mg/mL) in PBS with 100 mM sodium carbonate pH 8.3 were incubated with Alexa FluorTM 647 TFP-esterdye reagent (ThermoFisher Cat# A20173) at a dye:protein molar ratio of 10:1 for 1 hour at 25°C with mild agitation. The reactions were stopped, and removal of unincorporated excess dye was performed with 3 consecutive 10 mL ZebaTM (ThermoFisher cat# 89894) spin desalting columns pre-equilibrated with PBS pH 7.4. Purified samples were centrifuged for 10 minutes at 20,000 x g at 4°C, followed by quantification via absorption spectroscopy.
  • the degree of labeling (DOL) and concentration were based on absorbance readings at 280 nm and 650 nm using the equation below. Monomeric purity was determined using analytical HPLC Size-exclusion chromatography on a CytiviaTM SuperdexTM 200 Increase 5/150 GL column (Cytivia Cat# 28-9909-45) and PBS supplemented with 0.2 M arginine pH 7.2 as the running buffer.
  • Protein concentration (m) ([A28o-(Ad ye x CV280)] x dilution factor)/208000
  • Moles dye per mole protein (Ad ye x dilution factor)/(Sd ye x protein concentration (M) ) [00148] Where Edye (in cm -1 M' 1 ) is the approximate molar extinction coefficient of the Alexa FluorTM 647 dye (239,000).
  • Antibodies were also tested for stability at -80°C over 12 months using similar binding assay. Noting the shift in brightness of staining are due to changes in flow cytometer settings, whereas there was no significant change in apparent binding affinity for antibodies over the course of the 12 month test period. These results demonstrate that an AF-647 labelled antibody reagent can be stably stored for at least 12 months at -80°C.
  • Antibodies were also tested for use as a combined reagent in order to maximize the diversity of VHH proteins that can be identified with a labelling reagent.
  • cells stably expressing EGFR or CD22-specific sdAb-CAR proteins were stained with AF-647 labelled 3H12 antibody, 2A3 antibody, ora mixture of both antibodies. Results depicted in Fig. 8 show higher apparent affinity for the mixed antibody reagent, demonstrating that a mixed antibody product can be employed to label sdAb proteins on the surface of cells.
  • anti-VnH antibodies to detect the expression of sdAb in the context of other immunotherapy including but not limited to enumerating CAR expression on CAR modified NK cell therapy and to quantify titer of CAR expressing viral vectors has thus been demonstrated.
  • BCMA-sdAb proteins showed varying binding to BCMA- high and BCMA-low cells, and no apparent binding to BCMA-negative target cells.
  • MSLN-sdAb proteins displayed varying binding to cells.
  • a workflow as shown on Fig. 13 left was developed wherein tissue samples are stained with a purified sdAb, probed with anti-VnH secondary antibody, and finally detected using a commercial anti-mouse antibody staining reagent.
  • tissue samples are stained with a purified sdAb, probed with anti-VnH secondary antibody, and finally detected using a commercial anti-mouse antibody staining reagent.
  • a frozen tissue array with various human tissues was obtained and stained with a purified CD22- specific sdAb or a control antibody. These samples were then washed and stained with anti-VnH secondary antibody. Finally, slides were washed and stained with an anti-mouse detection reagent. As shown in Fig.
  • the native antigen-specific T-cell receptor molecules on human T cells are non- covalently associated on the cell surface with the CD3 molecular complex. Immobilization of this complex with surface-bound anti-CD3 monoclonal antibodies induces T cell activation without the need for antigen-specific recognition via the T cell receptor. Therefore, as expected, dose dependent activation of all T cells including un-transduced mock T cells and both sdAb and scFv-based CAR transduced T cells were observed in response to OKT3; the well-established T-cell activating anti-CD3 mAb which was used as the positive control (Fig. 14A).
  • the anti-VnH reagent used here specifically targets the sdAb domain of sdAb-CAR cells when immobilized on a 2D surface. Furthermore anti-VnH should not activate non-transduced T cells (mock) or single-chain variable fragment CAR-expressing cells (scFv-CAR). As expected, a dose dependent activation was only observed in sdAb-based CAR constructs with the anti-VnH mAb. No activation was seen in response to anti-VnH mAbs in untransduced T cells or scFv-based CAR-T cells (Fig. 14B).
  • Fig. 15 depicts the measure of cell expansion over time. Strong expansion of all 4 sdAb-based CAR-T cells were observed over time up to at least day 15 post exposure to the anti-VnH mAb. No such expansion was observed with the scFv-based CAR-T or un-transduced mock T cells suggesting anti-VnH mAb were capable of selectively expanding sdAb-based CAR constructs.
  • Fig 16 depicts the hypothetical mechanism by which an anti-VHH antibody might bind to a VHH and interrupt the cellular mechanisms required for activation of the CAR-expressing cells.
  • FIG. 17 depicts the results of a cell activation experiment demonstrating direct inhibition of an EGFR-targeted VHH-CAR by varying concentrations of anti-VHH antibodies alone or in combination.
  • the immortalized human T cell line, Jurkat was used for this experiment. These cells were engineered using lentiviral gene transfer to specifically express a VHH-CAR targeted towards the human EGFR receptor (as described in McComb et al, Front. Immunol., 21 July 2022, Vol. 13 (https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.864868).
  • EGFR-CAR cells were then placed at 37°C for 48 hours in co-culture with EGFR-positive SKOV3 human lung cancer cells, in the presence of decreasing concentrations of the anti-VHH antibodies, 2A3 and 3H12, alone or in combination, or vehicle control.
  • EGFR-CAR cells in the absence of target cells show low expression of the CD69 activation marker (open squares)
  • the addition of EGFR-positive target cell lines results in a strong upregulation of CD69 (open diamonds).
  • With increasing concentration of the anti-VHH antibodies alone or in combination we see a dose-dependent inhibition of the activation of EGFR-VHH-CAR cells in response to target cells.
  • antibody was conjugated to the microtube inhibitor mertansine (DM1) via the bifunctional cross-linker Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) using a 1-step synthesis method where N2'-Deacetyl-N2'-[3-[[1-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]- 2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]-maytansine (SMCC-DM1) with an active N- hydroxysuccinamide ester functional group was randomly reacted with an antibody surface accessible lysine primary amine group.
  • SMCC-DM1 with an active N- hydroxysuccinamide ester functional group was randomly reacted with an antibody surface accessible lysine primary amine group.
  • SMCC-DM1 solubilized in dimethyl acetamide was mixed with antibodies in 100 mM Sodium phosphate, 20 mM NaCI, 2mM EDTA pH 7.4, with a final DMA co-solvent concentration of 5% v/v and a SMCC-DM1 :antibody molar ratio of between 7 and 12 to 1. This mixture was incubated at room temperature overnight with no agitation. Conjugated antibody was purified using desalting columns into formulation buffer consisting of 20 mM Sodium succinate, 0.02% Polysorbate- 20 pH 6.0. Anti-VHH-DM1 antibody drug conjugate (ADC) was then purified using affinity column. This DM1 conjugated anti-VHH antibody is referred to as 3H12-DM1 below.
  • a cytotoxicity assay is then performed using 3H12-DM1 two human Jurkat T cell line, one without CAR expression (WT) and one with VHH-CAR expression. Varying concentrations of the 3H12-DM1 were mixed with the Jurkat cells and incubated the cells overnight. The next day, viability is assessed using flow cytometric assessment of cell condition. Fig. 18 shows a sharp drop in Jurkat cell viability for those cells that express the VHH-CAR, but no effect on WT Jurkat cells. These results demonstrate that Anti-VnH antibodies can be used effectively to specifically kill VHH-CAR expressing cells.
  • the anti-VnH reagent of the present invention may also be used to redirect the cytotoxicity or other cellular responses mediated by a VHH containing protein using a soluble VHH- redirector protein.
  • an scFv or other antibody derivative molecule would be linked to an additional antigen binding element, which could be scFv, sdAb, or another antigen targeting moiety. This would then effectively redirect responses against alternative targets.
  • This could be used for targeting multiple antigens on a single cell, for example, in the event that the first antigen targeted by the VHH containing protein would be downregulated or targeting alternative cells altogether.
  • Fig. 19 shows a diagram of this application.

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Abstract

The present document describes an anti-VHH antibody or antigen-binding fragment thereof that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), compositions comprising said antibodies, and uses and methods of use thereof. Compositions comprising more than 1 anti-VHH antibody are broadly reactive with VHH proteins providing a reagent for detecting VHHs generally. Methods for the detection of the surface expression of a sdAb domain from Lama glama on a cell, and methods of stimulating or blocking activation, expansion, or both of a chimeric antigen receptor (CAR)-T cell, using the antibody or antigen-binding fragment thereof are also disclosed.

Description

BROADLY REACTIVE ANTI-VHH ANTIBODIES
FIELD OF THE INVENTION
[0001] The present disclosure describes antibodies that specifically bind to the variable domain of Camelid (e.g. Ilama)-derived single domain antibodies (sdAbA/nH).
BACKGROUND
[0002] Chimeric Antigen Receptors (CAR) are used to redirect immune effector cells, such as T- or NK-cells, to specific targets, such as tumour-associated antigens. They consist of an antigen-binding sequence fused to immune cell activating domains which are expressed in T-, NK or other immune effector cells. The antigen binding sequence can be designed using modified monoclonal antibodies (single-chain Fv, orscFv) ormore recently, single-domain antibodies (sdAb’s or nH’s). In pre-clinical or clinical settings, it is important to detect the level of expression of the CAR transduced/transfected at the cell surface. The cell surface expression of scFv-based CAR molecules can be detected using fluorescently labelled commercially available secondary reagents such as anti-human or anti-mouse IgG (heavy and light) secondary antibodies, which have varying utility depending on the characteristics of the CAR being used. However, there are a limited number of commercial reagents available for the detection of single-domain antibodies based-CAR.
[0003] There are a few anti-camelid antibodies such as Genscript MonoRab™ Rabbit Anti- Camelid nH Cocktail (CatA02014), or Jackson polyclonal anti-Alpaca secondary antibodies or Rockland rabbit polyclonal anti-VnH. However, none of these have been demonstrated to be useful in flow cytometry to detect llama-derived CAR-sdAbs. Nor can they be used for modulating specific activation and expansion of CAR-sdAb expressing cells, or as a strategy for depletion of CAR-sdAb expressing cells.
[0004] Therefore, there is a need for a novel anti-VnH antibody that binds specifically to Camelid HH (e.g. the variable domain of a single domain antibody (sdAb) from Lama glama), particularly one that mitigates the shortcomings of existing compounds.
SUMMARY
[0005] As such, Applicants have generated mouse anti-camelid single domain antibody antibodies (herein referred to as anti-VnH antibodies) that can be used as a detector for llama derived sdAb-based therapeutics including, but not limited to, CAR or purified sdAbs. These could be used as reagents to detect llama-derived sdAb-based therapies across many applications including, but not limited to, ELISA, flow cytometry, immunofluorescence, and immunohistochemistry. Furthermore, additional applications of a high quality anti-sdAb antibody include the detection of Hama-sdAb-based CAR expression on immune cells and ex vivo or in vivo expansion/depletion, targeted depletion of CAR- expressing cells, or re-direction of such sdAb based cellular therapies.
[0006] Advantageously, such antibodies cross react with the relatively constant framework regions of the VHH protein; this means they have the desirable capacity to bind a wide variety of VHH proteins regardless of variable regions. Advantageously, such antibodies fail to cross react with human antibodies, preventing non-specific binding in cell and tissue staining applications.
[0007] The anti-VnH antibodies or antigen-binding fragments thereof of the present disclosure may be used, for example, for ex vivo or in vivo detection of a therapeutic comprising llama-derived VHH including but not limited to CAR-T therapeutics. Another application of these antibodies is the detection of camelid VHH proteins bound to cells or tissues for assessment of antigen expression on said cells or tissues or for assessing specificity of antibody binding. In addition, these antibodies can be used in certain formats to block such HH CAR activation, or in other formats for activation and expansion of VHH CAR- T consisting of target antigen binding domains consisting of Camelid VHH.
[0008] In one aspect, the present invention provides a composition comprising an anti-VHH antibody, said anti-VHH antibody comprising an antibody or antigen-binding fragment thereof, that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3), wherein composition comprises: a) a first anti-VHH antibody, said CDR H1 , H2, H3, L1 , L2, and L3 comprising the amino acid sequences: CDR H1 : GYGIS (SEQ ID NO:1), CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2), CDR H3: FYFELAY (SEQ ID NO:3), CDR L1 : RTNLGGNYMY (SEQ ID NO:4), CDR L2: YTSNLAP (SEQ ID NO:5), and CDR L3: QQFTSSASTWT (SEQ ID NO:6), respectively, in admixture with a diluent, excipient, or carrier; or b) a second anti-VHH antibody, said CDR H1 , H2, H3, L1 , L2, and L3 comprising the amino acid sequences: CDR H1 : NYHMS (SEQ ID NO:7), CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8), CDR H3: QRSDYWFDY (SEQ ID NO:9), CDR L1 : RASENIYSYLA (SEQ ID NO:10), CDR L2: NAKTLTE (SEQ ID NO:11), and CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively, in admixture with a diluent, excipient, or carrier; or c) both the first and the second anti-VHH antibody.
[0009] According to an embodiment, there is provided an anti-VHH antibody, comprising an antibody or antigen-binding fragment thereof, that binds specifically to a Camelid VHH (e.g. single domain antibody (sdAb) domain from Lama glama), wherein the antibody or antigen-binding fragment comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3), wherein said CDR H1 , H2, H3, L1 , L2, and L3 comprise an amino acid sequence comprising:
CDR H1 : GYGIS (SEQ ID NO:1),
CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
CDR H3: FYFELAY (SEQ ID NO:3),
CDR L1 : RTNLGGNYMY (SEQ ID NO:4),
CDR L2: YTSNLAP (SEQ ID NO:5), and
CDR L3: QQFTSSASTWT (SEQ ID NO:6), respectively; or
CDR H1 : NYHMS (SEQ ID NO:7),
CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8),
CDR H3: QRSDYWFDY (SEQ ID NO:9),
CDR L1 : RASENIYSYLA (SEQ ID NQ:10),
CDR L2: NAKTLTE (SEQ ID NO:11), and
CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively.
[0010] In one aspect, the present invention provides a composition comprising two antibodies, the first antibody comprising the following CDR sequences:
CDR H1 : GYGIS (SEQ ID NO:1),
CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
CDR H3: FYFELAY (SEQ ID NO:3),
CDR L1 : RTNLGGNYMY (SEQ ID NO:4),
CDR L2: YTSNLAP (SEQ ID NO:5), and
CDR L3: QQFTSSASTWT (SEQ ID NO:6), respectively; and the second antibody comprising the following CDR sequences:
CDR H1 : NYHMS (SEQ ID NO:7),
CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8),
CDR H3: QRSDYWFDY (SEQ ID NO:9),
CDR L1 : RASENIYSYLA (SEQ ID NQ:10),
CDR L2: NAKTLTE (SEQ ID NO:11), and
CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively.
[0011] This composition comprising two antibodies may be particularly advantageous, as it provides broadly reactive antibodies that will react with most or almost all HH molecules. This mixture of antibodies advantageously increases the breadth of reactivity, while preferably maintaining a high- quality defined product. Thus, in one advantageous embodiment, the present invention provides an off-the-shelf screening/detection reagent that is able to detect almost all VHHs, regardless of sequence or specificity. This is thought to be because the framework regions of different VHHs nevertheless have areas with similar or identical sequence identity. Use of two anti-VHHs advantageously provides even broader reactivity than a single anti-VHH antibody.
[0012] The anti-VHH antibody may further comprise four variable heavy domain framework regions (HFR)(HFR 1 , 2, 3 and 4), wherein said HFR 1 , 2, 3, and 4 comprise an amino acid sequence comprising:
HFR 1 : QAQLQQSGAELARPGASVRLSCKASGYTFT (SEQ ID NO:13),
HFR 2: WVKQRTGQGLEWIG (SEQ ID NO:14),
HFR 3: TATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO: 15), and
HFR 4: WGQGTLVTVSA (SEQ ID NO:16); or
HFR 1 : EVQLVESGGGLVQPGGSLKLSCAASGFSFS (SEQ ID NO:17),
HFR 2: WFRQTPEKRLEWV (SEQ ID NO:18),
HFR 3: GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:19), and
HFR 4: WGQGTTLTVSS (SEQ ID NQ:20).
[0013] The anti-VHH antibody may further comprise four variable light domain framework regions (LFR)(LFR 1 , 2, 3 and 4), wherein said LFR 1 , 2, 3, and 4 comprise an amino acid sequence comprising:
LFR 1 : EIVLTQFPAIISASLGEKVTMNC (SEQ ID NO:21),
LFR 2: WYQQKSDASPRLWIY (SEQ ID NO:22),
LFR 3: GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:23), and
LFR 4: FGGGTKLEIK (SEQ ID NO:24); or
LFR 1 : DIQMTQSPASLSASVGETVTITC (SEQ ID NO:25),
LFR 2: WFQQRQGKSPHLLVY (SEQ ID NO:26),
LFR 3: GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC (SEQ ID NO:27), and
LFR 4: FGSGTKLEIK (SEQ ID NO:28).
In one aspect, the present invention provides an anti-VHH antibody, consisting of a light chain only antigen-binding antibody fragment that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises an amino acid sequence comprising three variable light domain CDR (CDR L1 , L2 and L3), consisting of either:
CDR L1 : RTNLGGNYMY (SEQ ID N0:4),
CDR L2: YTSNLAP (SEQ ID N0:5), and
CDR L3: QQFTSSASTWT (SEQ ID N0:6), respectively; or
CDR L1 : RASENIYSYLA (SEQ ID NQ:10),
CDR L2: NAKTLTE (SEQ ID NO:11), and
CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively.
[0014] In one aspect, the present invention provides an anti-VHH antibody, consisting of a heavy chain only antigen-binding antibody fragment that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises an amino acid sequence comprising three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), consisting of either:
CDR H1 : GYGIS (SEQ ID NO:1),
CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
CDR H3: FYFELAY (SEQ ID NO:3), respectively; or
CDR H1 : NYHMS (SEQ ID NO:7),
CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8),
CDR H3: QRSDYWFDY (SEQ ID NO:9), respectively.
[0015] The anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising:
QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29).
[0016] The anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30).
[0017] The anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising:
EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31). [0018] The anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
[0019] The anti-VHH antibody may comprise a variable heavy domain ( H) comprising amino acid sequence comprising:
QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29), and a variable light domain ( L) comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
[0020] The anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30), and a variable light domain (VL) comprising amino acid sequence comprising:
DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
[0021] The antibody or antigen-binding fragment may be an IgA, IgD, IgE, IgG, or IgM.
[0022] The antigen-binding fragment may be a single-domain antibody (sdAb), or a singlechain variable fragment (scFv).
[0023] The sdAb may comprise three CDR (CDR1 , 2 and 3) comprising SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, respectively, or comprising SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.
[0024] The antibody or an antigen-binding fragment thereof may be humanized or partially humanized.
[0025] The antibody or antigen-binding fragment may be operable to stimulate or block activation, expansion, or both activation and expansion of an sdAb-based chimeric antigen receptor (CAR)-T cell.
[0026] According to another embodiment, the antibody or antigen-binding fragment is bound directly or through a linker to a functional moiety.
[0027] The antibody or antigen-binding fragment (anti-VHH) may be linked to the functional moiety via a peptide linker. The peptide linker may comprise 1-40 amino acid residues. The peptide linker may comprise the amino acid sequence (SS)n, (GGG)n, (GGGG)n, (GGGS)n, or (SSGGG)n, (SEQ ID NO: 33), or (GGGGS)n (SEQ ID NO: 34), wherein n > 1 .
[0028] The functional moiety may be a detectable label, a peptide, a polypeptide, a protein, an enzyme, a nucleic acid, a lipid particle, a cytotoxic agent, a drug, a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof; wherein the antibody or antigen-binding fragment is functional and wherein the linked detectable label, peptide, polypeptide, protein, enzyme, nucleic acid, lipid particle, cytotoxic agent, drug, second antibody, antibody fragment, second antigen-binding fragment, or the combination of any two or more thereof is functional.
[0029] The detectable label may be a fluorescent marker, a radioactive marker, an MRI contrast agent, a detectable secondary antibody, or combinations thereof.
[0030] According to another embodiment, there is provided a composition comprising the anti- VHH antibody of the present invention, and a pharmaceutically acceptable diluent, carrier or excipient.
[0031] According to another embodiment, there is provided a nucleic acid molecule encoding an anti-VHH antibody of the present invention.
[0032] According to another embodiment, there is provided a vector comprising the nucleic acid molecule of the present invention operably linked to one or more regulatory elements to allow expression of the antibody or antigen-binding fragment of the present invention in a host cell.
[0033] According to another embodiment, there is provided a cell comprising the vector of the present invention for expressing the anti-VHH antibody of the present invention.
[0034] According to another embodiment, there is provided a method for the detection of the surface presence of a sdAb domain from Lama glama on a cell comprising detecting an anti-VHH antibody of the present invention, or the compound of the present invention contacting said cell.
[0035] The anti-VHH antibody of the present invention, or the compound of the present invention may comprise a detectable label such as Alexa Fluor™ 647 shown in examples herewith.
[0036] The cell may be a chimeric antigen receptor (CAR)-T cell.
[0037] According to another embodiment, there is provided a method for the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell comprising a CAR comprising a sdAb domain from Lama glama, comprising contacting said CAR-T cell with an anti-VHH antibody of the present invention.
[0038] According to another embodiment, there is provided a method for redirection of a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR comprising a sdAb domain from Lama glama, comprising contacting said CAR-T cell with the compound of the present invention, the compound comprising a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof, specific for a second antigen different from the antigen for which the CAR is specific. In one aspect, the present invention provides an anti-VHH antibody operable
• to either stimulate or block the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell having a VHH on the surface;
• to detect a VHH on the surface a cell;
• to redirect a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR with a surface VHH; or
• to assess the binding properties of a VHH.
[0039] In one aspect, the present invention provides a method for the detection of a VHH on the surface a cell comprising detecting said VHH using the anti-VHH antibody described herein. In one aspect, the cell is a CAR-T cell. In one aspect, the anti-VHH antibody comprises a detectable label.
[0040] In one aspect, the present invention provides a method for either stimulating or blocking the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell having a VHH on the surface, comprising contacting said CAR-T cell with the the composition or anti-VHH antibody as described herein.
[0041] In one aspect, the present invention provides a method for redirection of a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR with a surface VHH, comprising contacting said CAR-T cell with the composition or anti-VHH antibody described herein, wherein the anti-VHH antibody comprises a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof, specific for a second antigen different from the antigen for which the CAR is specific.
[0042] In one aspect, the present invention provides a method for assessing the binding properties of a VHH, comprising subjecting a sample in vivo, ex vivo, or in vitro to purified VHH and then detecting binding of said VHH using the composition or anti-VHH antibody described herein.
[0043] In one aspect, the present invention provides a use of the anti-VHH antibody described herein to directly or indirectly detect VHH proteins as a method of assessing the binding properties of VHH proteins. [0044] In one aspect, the present invention provides an anti-VHH antibody or composition as described herein, wherein the anti-VHH antibody is operable to stimulate or block activation, expansion, or both activation and expansion of an sdAb-based chimeric antigen receptor (CAR)-T cell.
[0045] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
[0047] Fig. 1 illustrates binding of eight hybridoma supernatants against a panel of sdAb- based CARs Generated using 2 separate human donor T cells. Un-transduced T cells and two scFv- based CARs (CD22-m971 and CD19-FMC63) were used as controls.
[0048] Fig. 2 illustrates CAR expression detected by evaluating GFP expression versus binding of the anti-VnH mAb 2A3. Un-transduced T cells (Mock), CD19-scFv, and BCMA-scFv based CAR were used as negative controls, where no binding was expected.
[0049] Fig. 3 illustrates amino acid sequences of H and VL and CDR regions of anti-VnH antibodies 2A3 and 3H12.
[0050] Fig. 4 illustrates binding of hybridoma expressed versus recombinantly expressed mouse lgG2a formats of the anti-VHH monoclonal antibodies to three different llama-VHH proteins.
[0051] Fig. 5A illustrates detection of sdAb-based CAR expression on Jurkat T cells using unlabelled recombinant anti-VHH antibody 2A3, as well as the long-term stability of the anti-VHH antibody when stored at -80°C.
[0052] Fig. 5B illustrates detection of sdAb-based CAR expression on Jurkat T cells using unlabelled recombinant anti-VnH antibody 3H12 as well as the long-term stability of the anti-VnH antibodies when stored at -80°C.
[0053] Fig. 6 illustrates the results of hydrogen-deuterium exchange mass spectrometry experiment, which identifies the specific interaction peptides for the anti-VHH antibodies. This experiment reveals two partially overlapping, but distinct for the 2A3 and 3H12 antibodies. [0054] Fig 7. Illustrates the strength of binding for the 2A3 and 3H12 anti-VHH antibodies individually or in a 1 :1 cocktail against 76 unique VHH proteins, as assessed via ELISA. These results reveal negative binding of an antibody mixture for only 1 of 76 VHH proteins, with the mixture consistently outperforming the binding of either antibody alone.
[0055] Fig. 8 illustrates the comparability of two different batches of Alexa Fluor™ (AF)-467 labelled anti-VHH antibodies for their ability to detect expression of sdAb-based CAR constructs on T cells. This figure also demonstrates that a mixture of two anti-VnH antibodies (3H12 and 2A3) can be effectively employed to stain cells with surface expression of sdAb-CARs.
[0056] Fig. 9 illustrates comparability of sdAb-based CAR detection using AF-647-conjugated F233-2A3-2 (left panel) or FITC-conjugated CD22 protein (ACRO BioSystems) (right panel). For this work, healthy donor enriched CD4/CD8 lymphocytes that were 1-day post-activation with TransAct (10 pl/106 cells) were transduced with sdAb-based CAR. Transduction was performed in 4x24-well plates (at 1x106 cells/well) then expanded in Grex flasks to ~650 x106 cells at day 14. Cells were then stained with AF-647-conjugated F233-2A3-2 or FITC-conjugated CD22 protein (ACRO).
[0057] Fig. 10 illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of CD22-specific 1 ug13 and 1 ug36 to CD22-expressing Ramos cells but not CD22-knockout Ramos cells is shown. A negative control sdAb specific for an irrelevant bacterial protein (B131) was also used to demonstrate the specificity of sdAb binding.
[0058] Fig. 11 further illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of BCMA- specific sdAbs to BCMA-high RPMI8226 cells, BCMA-low Raji cells, or BCMA-negative Jurkat cells is shown.
[0059] Fig. 12 further illustrates the application of anti-VnH antibodies as secondary labelling reagents to assess cell binding of soluble purified sdAb proteins. Specifically the binding of mesothelin- specific sdAbs to mesothelin-high H292 cells is shown.
[0060] Fig 13. illustrates the application of anti-VnH antibodies as secondary labelling reagent in tissue and cell level binding in an immunohistochemical assessment of sdAb specificity. Specifically, a frozen tissue microarray was stained with either CD22-specific sdAb or an irrelevant specificity control sdAb, followed by staining with secondary anti-VHH, and eventually a tertiary anti-mouse detection reagent. Staining results for representative tissues are shown here.
[0061] Fig. 14A illustrates the activation of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells by anti-CD3 antibody OKT3. [0062] Fig. 14B illustrates the activation of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells by anti-VnH monoclonal antibody 3H12.
[0063] Fig. 15 illustrates an expansion of untransduced primary human T (Mock) or CAR-T (scFv or sdAb-based CARs) cells following simulation with surface immobilized anti-VnH monoclonal antibody 3H12. All anti-VnH stimulated sdCAR cells expanded for at least 15 days, with only transient expansion on scFvCAR or untransduced T cells due to previous polyclonal activation.
[0064] Fig 16 illustrates a diagram of the potential mode by which a soluble anti-VHH antibody might interfere with and inhibit CAR activation.
[0065] Fig 17. Illustrates the inhibition of CAR reactivity in the presence of various doses of 2A3 or 3H12 anti-VHH antibodies, or a combination thereof. Specifically, Jurkat cells engineered to express an EGFR-directed CAR were combined with EGFR-expressing CAR cells, with varying doses of the anti-VHH antibodies, a mixture of the antibodies, or vehicle control.
[0066] Fig. 18 illustrates the loss in viability observed following treatment of VHH-CAR expressing Jurkat cells but not WT Jurkat cells following treatment with anti-VHH antibody drug conjugate.
[0067] Fig. 19 illustrates a molecular strategy wherein the anti-VHH molecule is used to redirect the CAR towards additional target molecules.
[0068] It will be noted that throughout the appended drawings, like features are identified by like reference numerals.
DETAILED DESCRIPTION
[0069] The present invention is directed to a technology for the specific recognition of the variable domain of a single domain antibody (sdAb) from Lama glama. In embodiments there is disclosed antibody or antigen-binding fragments thereof, identified herein as anti-VHH, that bind specifically to single domain antibody (sdAb or VHH) domains from Lama glama. that the terms sdAb and VHH are used interchangeably herein, unless specifically defined otherwise.
[0070] In one aspect, the anti-VHH antibody or antigen-binding fragment comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3).
[0071] The CDR H1 , H2, H3, L1 , L2, and L3 of the invention may comprise the following amino acid sequence comprising: 1) CDR H1 : GYGIS (SEQ ID N0:1), CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID N0:2), and CDR H3: FYFELAY (SEQ ID N0:3); and CDR L1 : RTNLGGNYMY (SEQ ID N0:4), CDR L2: YTSNLAP (SEQ ID N0:5), and CDR L3: QQFTSSASTWT (SEQ ID N0:6), respectively; or or
2) CDR H1 : NYHMS (SEQ ID NO:7), CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8), and CDR H3: QRSDYWFDY (SEQ ID NO:9); and CDR L1 : RASENIYSYLA (SEQ ID NQ:10), CDR L2: NAKTLTE (SEQ ID NO:11), and CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively.
[0072] According to embodiments, the anti-VHH antibody may further comprising fourvariable heavy domain framework regions (HFR)(HFR 1 , 2, 3 and 4). The HFR 1 , 2, 3, and 4 may comprise the following amino acid sequence comprising:
1) HFR 1 : QAQLQQSGAELARPGASVRLSCKASGYTFT (SEQ ID NO:13), HFR 2: WVKQRTGQGLEWIG (SEQ ID NO:14), HFR 3: TATLTADKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO:15), and HFR 4: WGQGTLVTVSA (SEQ ID NO: 16). or
2) HFR 1 : EVQLVESGGGLVQPGGSLKLSCAASGFSFS (SEQ ID NO:17), HFR 2:
WFRQTPEKRLEWV (SEQ ID NO:18), HFR 3:
GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:19), and HFR 4: WGQGTTLTVSS (SEQ ID NQ:20).
[0073] According to embodiments, the anti-VHH antibody may further comprising fourvariable light domain framework regions (LFR)(LFR 1 , 2, 3 and 4). The LFR 1 , 2, 3, and 4 may comprise the following amino acid sequence comprising:
1) LFR 1 : EIVLTQFPAIISASLGEKVTMNC (SEQ ID NO:21), LFR 2: WYQQKSDASPRLWIY (SEQ ID NO:22), LFR 3: GVPARFSGSGSGNSYSLTISSMEGEDAATYYC (SEQ ID NO:23), and LFR 4: FGGGTKLEIK (SEQ ID NO:24); or
2) LFR 1 : DIQMTQSPASLSASVGETVTITC (SEQ ID NO:25), LFR 2: WFQQRQGKSPHLLVY (SEQ ID NO:26), LFR 3: GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC (SEQ ID NO:27), and LFR 4: FGSGTKLEIK (SEQ ID NO:28).
[0074] According to embodiments, the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29). [0075] According to embodiments, the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NO:30).
[0076] According to embodiments, the anti-VHH antibody may comprise a variable light domain (V ) comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
[0077] According to embodiments, the anti-VHH antibody may comprise a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
[0078] According to embodiments, the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29), and a variable light domain (V ) comprising amino acid sequence comprising: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID NO:31).
[0079] According to embodiments, the anti-VHH antibody may comprise a variable heavy domain (VH) comprising amino acid sequence comprising: EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NQ:30), and a variable light domain (VL) comprising amino acid sequence comprising: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
[0080] According to an embodiment of the present invention, the anti-VHH antibody may be single-domain antibody (sdAb), an engineered single-domain fragment, a fragment antigen-binding (Fab), a single-chain variable fragment (scFv), or a single-chain fragment antigen-binding (scFab). The antibody or antigen-binding fragment may be an IgA, an IgD, an IgE, an IgG, or an IgM.
[0081] According to embodiments, the sdAb may comprises three CDR (CDR1 , 2 and 3) comprising SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3, respectively, or comprising SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, that binds specifically to a single domain antibody (sdAb) domain from Lama glama. [0082] As used herein, the expression “substantially identical sequence” is intended to mean an amino acid sequence which may comprise one or more conservative amino acid mutations. It is known in the art that the introduction of one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physicochemical or functional properties compared to the reference sequence. In such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity). According to one embodiment, one or more conservative amino acid mutations may be made to the one or more framework regions of the anti-VHH antibody while maintaining both the CDR sequences and the overall structure of the CDR of the antibody or antigen-binding fragment; thus the specificity and binding of the antibody or antigen-binding fragment are maintained. According to another embodiment, one or more conservative amino acid mutations may be made to the one or more framework regions of the anti-VHH antibody and to a CDR sequence while maintaining the antigen-binding function of the overall structure of the CDR of the antibody or antigen-binding fragment; thus the specificity and binding of the antibody or antigen-binding fragment are maintained.
[0083] In a non-limiting example, a conservative mutation may be a conservative amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another amino acid of the same group. By the term “basic amino acid” it is meant a hydrophilic amino acid having a side chain pK value of greater than 7, which is typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q). By the term “hydrophobic amino acid” (also “non-polar amino acid”) it is meant an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Vai or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refers to a hydrophilic amino acid having a side chain pK value of less than 7, which is typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E) and aspartate (Asp or D).
[0084] Sequence identity is used to evaluate the similarity of two sequences. It is determined by calculating the percentage of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.
[0085] The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or any percentage there between, at the amino acid level to sequences described herein. Importantly, a substantially identical sequence retains the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to one or more conservative amino acid mutations. In a non-limiting example, the present invention may be directed to an antibody or antigen-binding fragment comprising a sequence at least at least 95%, at least 98%, or at least 99% identical to that of one or more of the antibodies or antigen-binding fragments described herein.
[0086] The term “antibody”, also referred to in the art as “immunoglobulin” (Ig), as used herein refers to an antigen-binding protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while the heavy chain folds into a variable (VH) and three constant (CH1 , CH2, CH3) domains. Interaction of the heavy and light chain variable domains (VH and L) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.
[0087] The light and heavy chain variable regions are responsible for binding a target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (VH) and light (VL) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat and Wu (1991) define the “complementarity-determining regions” (CDRs) based on sequence variability at the antigen-binding regions of the VH and VL domains. Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the VH and L domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping. Those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. The CDRs/loops are identified herein according to the IMGT nomenclature scheme (i.e., CDR1 , 2 and 3, for each variable region).
[0088] An “antibody fragment” or “antigen-binding fragment” as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or it may be a non-naturally occurring antibody fragment obtained, for example, by manipulation of a naturally-occurring antibody or by recombinant methods. For example, an antibody fragment may include, but is not limited to, a Fv, a single-chain Fv (scFv; a molecule consisting of VL and VH connected with a peptide linker), a Fab, a F(ab’)2, single-domain antibody (sdAb; a fragment composed of a single VL or VH or a VHH), or a multivalent presentation of any of these. Antibody fragments such as those just described may require one or more linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches for their construction.
[0089] As used herein the term “anti-VHH antibody” comprises both “antibodies” (such as monoclonal antibodies) as well as “antigen-binding fragments”, unless specified otherwise.
[0090] In a non-limiting example, the antigen-binding fragment of the present invention may be a sdAb derived from a naturally-occurring source. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed VHH. SdAbs have also been observed in shark and are termed VNAR (Nuttall et al, 2003). Other sdAbs may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term “sdAb” includes an sdAb directly isolated from a VH, VHH, VL, OTVNAR reservoir of any origin through phage display or other technology, an sdAb derived from the aforementioned sdAb, a recombinantly produced sdAb, as well as an sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.
[0091] SdAbsA/nHs possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997). They can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of one or more non-canonical disulfide bonds (Hussack et al, 2011a, b; Kim et al, 2012), may also be brought to the sdAb.
[0092] A person of skill in the art would be well-acquainted with the structure of a singledomain antibody/ VHHS (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDRs may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDRs may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDRs of the sdAb, VHHS or variable domain are referred to herein as CDR1 , CDR2, and CDR3.
[0093] The present invention further encompasses an antibody or an antigen-binding fragment that is “humanized” using any suitable method known in the art, such as, but not limited to, CDR grafting or veneering. Humanization of an antibody or an antigen-binding fragment comprises replacing an amino acid in the antibody or antigen-binding fragment sequence with its human counterpart, as found in the human consensus sequence, without substantial loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or antigen-binding fragment when introduced into a human subject. In the process of CDR grafting, one or more than one of the CDRs defined herein may be fused or grafted to a human variable region (VH, orVi.), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to a human antibody fragment framework region (Fv, scFv, Fab) or to another protein of similar size and nature onto which a CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of the one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the antibody or antigen-binding fragment for its target (i.e ., a human/rhesus/rat/mouse TGF-p family member, collectively referred to as TGF-p family member) is likely minimally affected. CDR grafting is known in the art and is described in at least the following: US Patent No. 6180370, US Patent No. 5693761 , US Patent No. 6054297, US Patent No. 5859205, and European Patent No. 626390. Veneering, also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of an antibody or antigen-binding fragment; thus, preserving buried nonhumanized residues, which may be important for CDR conformation, while minimizing the potential for immunological reaction against solvent-exposed regions. Veneering is known in the art and is described in at least the following: US Patent No. 5869619, US Patent No. 5766886, US Patent No. 5821123, and European Patent No. 519596. Persons of skill in the art would also be amply familiar with methods of preparing such humanized antibody fragments and humanizing amino acid positions. [0094] According to an embodiment, the antibody or antigen-binding fragment of the present invention may be operable to stimulate activation, expansion, or both activation and expansion of an sdAb chimeric antigen receptor (CAR)-T cell.
[0095] The antibody or antigen-binding fragment according to the present invention may comprise one or more additional sequences to aid in expression, detection or purification of the antibody or antigen-binding fragment. Any such sequence or tag known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or antigen-binding fragment may comprise a targeting or signal sequence (such as, but not limited to, ompA or pelB), a detection/purification tag (such as, but not limited to, c-Myc, HA, His5, or His6), or a combination of any two or more thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al. in WO 95/04069 or by Voges et al. in WO/2004/076670. As is also known to those of skill in the art, a linker sequence may be used in conjunction with the additional sequence or tag, or may serve as a detection/purification tag.
[0096] In another embodiment, there is disclosed a compound comprising an antibody or antigen-binding fragment according to the present invention, linked to a functional moiety, directly or via a linker sequence. As used herein, the term “linker sequence” is intended to mean a short (typically 40 amino acids or fewer) peptide sequence that is introduced between protein domains. Linker sequences are often composed of flexible residues such as glycine and serine so that the linked protein domains are free to move relative to one another. The linker sequence can be any linker sequence known in the art that would allow for the antibody and the functional moiety of the present invention to be operably linked for the desired function. The linker may be any sequence known in the art (either a natural or synthetic linker) that allows for an operable fusion comprising an antibody or antigen-binding fragment linked to a polypeptide (e.g., the functional moiety). For example, the linker sequence may be a linker sequence L such as (SS)n, (GGG)n, (GGGG)n, (GGGS)n, (SSGGG)n, (SEQ ID NO: 33), or (GGGGS)n (SEQ ID NO: 34) wherein n is equal to or greater than 1 , or from about 1 to about 5, or from about 1 to 15; or n may be any number that would allow for the operability of the compound of the present invention. In another example, the linker may be an amino acid sequence, for example, an amino acid sequence that comprises about 1 to about 40 amino acids, or about 3 to about 40 amino acids, or about 5 to about 40 amino acids, or about 10 to about 40 amino acids, or about 15 to about 40 amino acids, or about 20 to about 40 amino acids, or about 25 to about 40 amino acids, or about 30 to about 40 amino acids, or about 35 to about 40 amino acids, or about 3 to about 35 amino acids, or about 5 to about 35 amino acids, or about 10 to about 35 amino acids, or about 15 to about 35 amino acids, or about 20 to about 35 amino acids, or about 25 to about 35 amino acids, or about 30 to about 35 amino acids, or about 3 to about 30 amino acids, or about 5 to about 30 amino acids, or about 10 to about 30 amino acids, or about 15 to about 30 amino acids, or about 20 to about 30 amino acids, or about 25 to about 30 amino acids, or about 3 to about 25 amino acids, or about 5 to about 25 amino acids, or about 10 to about 25 amino acids, or about 15 to about 25 amino acids, or about 20 to about 25 amino acids, or about 3 to about 20 amino acids, or about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or about 15 to about 20 amino acids, or about 3 to about 15 amino acids, or about 5 to about 15 amino acids, or about 10 to about 15 amino acids, or about 15 to about 20 amino acids, or about 3 to about 10 amino acids, or about 5 to about 10 amino acids, or about 3 to about 5 amino acids, or up to 3, up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, or up to 40 amino acids.
[0097] As used herein, the term “functional moiety” is intended to mean a part of the compound having an activity, purpose, or task; relating to the way in which the compound is intended to work or operate. In embodiments, the functional moiety may be linked to the antibody or antigen-binding fragment, for example, through a chemical link pursuant to a chemical reaction, and/or through fusion of the antibody or antigen-binding fragment with the functional moiety, obtained for example using recombinant DNA technology, or through a linker sequence.
[0098] According to an embodiment, the antibody or antigen-binding fragment of the compound may be covalently bound directly or through a linker sequence to a detectable label (e.g., a fluorescent marker, for example Alexa Fluor™ 647), a radioactive marker, an MRI contrast agent, a detectable secondary antibody, or combinations thereof, to co-localize the therapeutic agent of the present invention to an anatomical location where the sdAb-CAR modified immune cells would home to, and for detection of sdAb-CAR modified immune cells), a peptide, a polypeptide (e.g. growth factor CIBP2, an antimicrobial cyclic peptide), a protein, an enzyme [such as iduronate-2-sulfatase (IDS), acid beta-glucosidase (GCase), a serine protease, a growth factor, etc.], another (or the same) antibody or a fragment operable to bind a target epitope (e.g. an anti-microbial antibody, an anti-inflammatory antibody, an intrabody, a BBB-crossing antibody, a neurodegeneration target antibody, an ion channel targeting antibody, a cancer associated antigen antibody, a checkpoint inhibitor targeting antibody, or a GPCR targeting antibody)(for any use and for example for use in imaging, diagnostic, affinity purification, etc.), a nucleic acid (e.g., an siRNA, an mRNAs, an aptamer, etc.), a lipid particle (e.g., a lipid micro- or nano- particle loaded with a given payload, such as a peptide, a polypeptide, a compound, a nucleic acid, etc.), a cytotoxic agent (i.e ., for elimination of sdAb-based CAR modified immune cells in the event of undesirable clinical outcome) or a combination of any two or more thereof, in which both the antibody or antigen-binding fragment and the rest of the compound (i.e., the functional moiety) remain functional for their intended purpose. In a preferred embodiment, the compound may be fused or linked to a second antibody or antigen-binding fragment, operable to bind a target epitope, which may be the same as, or distinct from the epitope of the antibody or antigen-binding fragment of the present invention. [0099] The antibody or antigen-binding fragment of the present invention may also be in a multivalent display format, also referred to herein as multivalent presentation. Multimerization may be achieved by any suitable method known in the art. For example, and without wishing to be limiting in any manner, multimerization may be achieved using self-assembly molecules such as those described in Zhang et al (2004a; 2004b) and W02003/046560, where pentabodies are produced by expressing a fusion protein comprising the antibody or antigen-binding fragment of the present invention and the pentamerization domain of the B-subunit of an AB5 toxin family (Merritt & Hol, 1995). A multimer may also be formed using the multimerization domains described by Zhu et al. (2010); this form, referred to herein as a “combody” form, is a fusion of the antibody or fragment of the present invention with a coiled-coil peptide resulting in a multimeric molecule (Zhu et al., 2010). Other forms of multivalent display are also encompassed by the present invention. For example, and without wishing to be limiting, the antibody or antigen-binding fragment may be presented as a dimer, a trimer, or any other suitable oligomer. This may be achieved by methods known in the art (Spiess et al, 2015), for example by direct linking connection (Nielsen et al, 2000), c-jun/Fos interaction (de Kruif & Logtenberg, 1996), or “Knob into holes” interaction (Ridgway et al, 1996).
[00100] Another method known in the art for multimerization is to dimerize the antibody or antigen-binding fragment using an Fc domain, such as, but not limited to a human Fc domain. The Fc domain may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to lgG1 , lgG2, etc. In this approach, the Fc gene is inserted into a vector along with the sdAb gene to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed, then purified. For example, and without wishing to be limiting in any manner, a multivalent display format may encompass a chimeric or humanized format of VHH of the present invention linked to an Fc domain, or bi or tri-specific antibody fusions with two or three VHHS recognizing unique epitopes. Such antibodies are easy to engineer and produce, can greatly extend the serum half-life of a sdAb, and may be excellent tumor imaging reagents (Bell et al., 2010).
[00101] The Fc domain in the multimeric complex as just described may be any suitable Fc fragment known in the art. The Fc fragment may be from any suitable source; for example, the Fc fragment may be of mouse or human origin. In a specific, non-limiting example, the Fc fragment may be a mouse Fc2b fragment or a human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010). The Fc fragment may be fused to the N-terminal or C-terminal end of the VHH or humanized version of the present invention.
[00102] Each subunit of the multimers described above may comprise the same or different antibodies or antigen-binding fragments of the present invention, which may have the same ordifferent specificity. Additionally, the multimerization domains may be linked to the antibody or antigen-binding fragment using a linker, as required; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules but should not hamper the antigenbinding properties of the antibody or antigen-binding fragment. As defined above, the linker sequence can be any linker known in the art that would allow for the compound of the present invention to be prepared and be operable for the desired function.
[00103] According to another embodiment, the present invention also encompasses a composition comprising one or more than one anti-VHH antibody of the present invention and/or compound of the present invention as described herein. The composition may comprise a single anti- VHH antibody and/or compound as described above, or the composition may comprise a mixture of anti-VHH antibody and/or compounds. Furthermore, in a composition comprising a mixture of anti-VHH antibody and/or compounds of the present invention, the anti-VHH antibody and/or compounds may have the same specificity, or they may differ in their specificities; for example, and without wishing to be limiting in any manner, the composition may comprise anti-VHH antibody and/or compounds specific to a single domain antibody domain of Lama glama (same or different epitope).
[00104] A composition according to the invention may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art that is compatible with other ingredients in the composition, that is compatible with the method of delivery of the composition, and that is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (such as, but not limited to, lyophilised or encapsulated), capsule form or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, or a suitable buffer, and optionally comprise one or more additives to improve solubility and/or stability. Reconstitution to produce a suspension may be effected in a buffer at a suitable pH to ensure the viability of the antibody orantigen- binding fragment. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be formulated for delivery of the antibody or antigen-binding fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time release, or other suitable technologies for delivery of the anti-VHH antibody and/or compound of the present invention. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present sdAb and/or compound.
[00105] The invention also encompasses a nucleic acid molecule comprising a nucleotide sequence encoding an antibody, antigen-binding fragment, or compound of the present invention. The invention further comprises a vector comprising the nucleic acid molecule; a cell comprising the vector, for expressing the antibody, antigen-binding fragment, or compound of the present invention, and a cell for expressing the antibody, antigen-binding fragment, or compound of the present invention.
[00106] According to another embodiment, there is provided a method for the detection of the surface expression of a sdAb domain from Lama glama on a cell comprising detecting the anti-VHH antibody of the present invention, the compound of the present invention, contacting the cell. The anti- VHH antibody, or the compound may comprises a detectable label which is Alexa Fluor™ 467. According to an embodiment, the cell may be a chimeric antigen receptor (CAR)-T cell.
[00107] According to another embodiment, there is provided a method for the use of an anti- VHH antibody or a mixture of anti-VHH antibodies in immobilized format either through passive abdsorption on a planar surface, direct chemical conjugation to a substrate, or other method. Such immobilized anti-VHH product can be applied to specific purification, activation, and/or expansion of a chimeric antigen receptor (CAR)-T cell comprising a chimeric antigen receptor incorporating a sdAb domain from Lama glama as the antigen-binding fragment thereof, or a compound of the present invention.
[00108] According to another embodiment, there is provided a method for the use of a solubilized anti-VHH antibody or a mixture of anti-VHH antibodies to inhibit the function of a chimeric antigen receptor incorporating a sdAb domain from Lama glama as the antigen-binding fragment thereof. Specifically, a soluble anti-VHH antibody or anti-VHH antibody mix can be applied to interfere with the interaction of such a sdAb-CAR molecule with its cognate antigen target and/or block subsequent cellular activation processes leading to a diminishment of CAR-mediated cellular response.
[00109] According to another embodiment, there is provided a method for the use of an anti- VHH antibody ora mixture of anti-VHH antibodies which have been chemically conjugated to a desired payload molecule for targeted delivery to a cell which expresses a chimeric antigen receptor incorporating a sdAb domain from Lama glama as the antigen-binding fragment thereof. The payload molecule could be, but is not limited to, molecules that are chemicals, radiochemicals, nucleotides, nucleic acids, lipid nanoparticles (LNP),, peptides, or proteins, intended to achieve a desired biological effect within targeted VHH-CAR expressing cells. This would include drugs, like radiopharmaceuticals and cytotoxic drugs.
[00110] In one aspect, the payload could be a cytotoxic molecule. According to another embodiment, there is provided a method for the use of a solubilized anti-VHH antibody or a mixture of anti-VHH antibodies which have been chemically conjugated to a cytotoxic payload to create an antibody-drug conjugate molecule for targeted delivery to a cell which expresses a chimeric antigen receptor incorporating a sdAb domain from Lama glama as the antigen-binding fragment thereof. A therapeutic incorporating an anti-VHH antibody drug conjugate molecule could be used to specifically deplete VHH-CAR expressing cells within a patient treated with a VHH-CAR cellular therapy, should toxicity or other side-effects of VHH-CAR therapy arise within a patient.
[00111] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE 1
GENERATION OF SDAB/VHH SPECIFIC MONOCLONAL ANTIBODIES
[00112] Monoclonal antibodies (mAb) against sdAbs were generated by immunizing mice with a purified recombinant sdAb, FC5-MOD (SEQ ID NO: 35).
Immunizations
[00113] Four six-week old female A/J mice (The Jackson Laboratory, Bar Harbor, ME) were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 pg of sdAb FC5- MOD antigen emulsified in Titermax adjuvant (Cedarlane Labs, Burlington, ON) at day 0 and in PBS without adjuvant at day 26. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) at day 33, and serum was stored at -20°C until further use.
ELISA (serum titer determination)
[00114] Pre- and post-immune sera titer of animals immunized with FC5-MOD antigen were assessed by ELISA. Unless otherwise stated, all incubations were performed at room temperature. Briefly, half-area 96-well plates (Costar #3690) were coated with 25 pl per well of FC5-MOD at 5 pg/ml in PBS and incubated overnight at 4°C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1 % bovine serum albumin (BSA, Sigma Cat#A7030). Blocking buffer was removed and 25 pl of serial dilutions of sera samples were added. After a 2 h incubation, microplates were washed 4 times with PBS-Tween 200.05% and 25 pl of a 1/5.000 dilution of alkaline phosphatase conjugated goat anti-mouse IgG (H+L) (#115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1 h incubation, microplates were washed 4 times and 25 pl of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for 30 min. Absorbance was read at 405 nm using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). All pre-immune bleeds were negative and all post-immune bleeds were very strong (above 1/12800) on immunogen.
Generation of hybridomas [00115] After 2-3 months, an i.p. booster injection (100 pg of FC5-MOD protein in PBS) was done 3 days prior to fusion experiment.
[00116] Fusion of the harvested spleen cells. All manipulations were done under sterile conditions. Spleen cells were harvested in Iscove’s Modified Dulbecco’s medium (IMDM, Gibco Cat. #31980-030) and fused to NS0 myeloma cell line using polyethylene glycol. Spleen cells and myeloma cells were washed in IMDM, counted in RBC lysing buffer (Sigma, Cat#7757-100ML) and mixed together at a 5:1 ratio. Pelleted cells were fused together by adding 1 ml of a 50% solution of PEG 4000 (EMD-Millipore Cat#9727-2) in PBS preheated at 37°C drop-wise over one minute, and incubated at 37°C for an additional 90 sec. The reaction was stopped by addition of 30 ml of IMDM at 22°C over a period of 2 min. After a 10 min incubation, freshly fused cells were spun at 233g for 10min. Cells were washed once in IMDM supplemented with 10% heat inactivated FBS (Sigma Cat #F1051 ).
[00117] Following fusion, cells were suspended ata concentration of 2X105 input myeloma cells per ml in HAT selection medium (IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat#P7539), 1 ng/ml mouse IL-6 (Biolegend Cat#575706), HAT media supplement (Sigma Cat#H0262) and L-glutamine (Hy-Clone Cat#SH30034.01) and incubated at 37°C, 5% CO2. The next day, hybridoma cells were washed and suspended at a concentration of 2-3X105 input myeloma cells per ml in semi-solid medium D (StemCell Technologies Cat. No. 03804) supplemented with 5% heat inactivated FBS, 1 ng/ml mouse IL-6 and 10 pg/ml FITC- F(ab’)2 Goat anti-mouse IgG (Jackson # 115- 096-071). The cell mixture was plated in Omnitray dish (Nunc cat#242811) and further incubated for 6-7 days at 37°C, 5% CO2. Fluorescent secretor clones were then transferred using a mammalian cell clone picker (ClonepixFL, Molecular Devices) into sterile 96-well plates (Costar #3595) containing 200 pl of IMDM supplemented with 20% heat inactivated FBS, penicillin-streptomycin, 1 ng/ml mouse IL-6, HT media supplement (Sigma Cat# H0137) and L-glutamine and incubated for 2-3 days at 37°C, 5% CO2.
[00118] Hybridoma supernatants were screened by ELISA to detect specific binders. To this end, 96-well half-area plates (Costar #3690) were coated with 25 pl of FC5-MOD or FC5 derivatives FC5-VHH or FC5-hFc-1X0, or P257 (unrelated sdAb) or EG2-hFc-X2 (unrelated sdAb) or BSA (negative control) at 5 pg/ml in PBS and incubated overnight at 4°C. Microplates were washed 3 times with PBS, blocked with PBS-BSA 1%, and 25 pl of hybridoma supernatants were added and incubated at 37°C, 5% CO2 for 2 hours. Plates were washed 4 times with PBS-Tween 20 0.05% and incubated for one hourat 37°C, 5% CO2 with 25 pl of secondary antibody alkaline phosphatase conjugated F(ab’)2 goat anti-mouse IgG Fc-gamma specific (Jackson Immunoresearch # 115-056-071) diluted 1/3000 in blocking buffer. After 4 washes with PBS-Tween 20 0.05%, 25 pl of a 1 mg/ml pNPP substrate solution was added and further incubated for one hour at 37°C. OD405nm measurements were done using a microplate reader (Spectramax® 340 PC, Molecular Devices®). [00119] From the F233 fusion of the mouse spleen cells, 8 mAbs pan-specific for sdAbs were identified (Table 1) from which supernatant was collected and evaluated for binding to other sdAbs and CAR-T cells.
Table 1. ELISA results of selected pan-specific anti-sdAbs mAbs clones
Characterization by ELISA on other sdAbs
[00120] Hybridoma supernatants were further analyzed by ELISA for positive reactivity to various sdAb proteins: FC5-MOD (immunogen), sdAb-1 ug13, and sdAb-1 ug36; and for negative reactivity to human antibodies using protX-hlgG (negative control). All 8 mAbs were found positive for sdAb-1 ug13 and -1 ug36 as well as for the original immunogen (positive control) and negative for protX- hlgG antigen (Table 2).
Table 2. ELISA results of selected pan-specific anti-sdAbs.
EXAMPLE 2
EVALUATION OF BINDING ON SINGLE-DOMAIN DERIVED CAR-T Characterization by flow cytometry on various single-domain-derived CAR-T
[00121] A panel of eight hybridoma supernatants were selected based on Jurkat-CAR binding results to assess their ability to bind human primary T cell derived sdAb-based CARs by flow cytometry. Hybridoma supernatants were normalized by antibody concentration. Primary CAR-T cells generated using T cells from 2 separate donors were incubated with the hybridoma supernatants, washed, and incubated with AF-647 anti-mAb Fab 2° antibody. Fig. 1 shows the binding of the eight hybridoma across 5 different sdAb-based CAR; all targeting human CD22. Untransduced T cells and two scFv- based CARs (CD22-m971 and CD19-FMC63) were used as controls.
[00122] Based on high specific binding across a panel of sdAb-based CARs, two hybridoma; F233-2A3 and F233-3H12 were re-cloned by limiting dilution to ensure their monoclonality. Their respective subclass was determined using IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit. Both were found to be of mouse lgG1 subclass.
EXAMPLE 3
EVALUATION OF BINDING ON PURIFIED ANTIGENS
Characterization by ELISA on denatured antigens
[00123] To evaluate if monoclonal antibodies bind to a conformational epitope, an ELISA analysis for selected anti-sdAb mAbs F233-2A3 and F233-3H12 was performed on native and denatured sdAbs.
[00124] Antigens at 0.3-1.2 mg/ml were incubated at 95°C for 5 min in PBS containing DTT at a final concentration of 40 mM. They were then incubated on ice for 5 min and diluted at their final coating concentration (5 pg/ml) for ELISA purpose.
[00125] Table 3 shows ELISA results (mean of n=2) of different mAb clones assessed on purified sdAbs, in native or denatured conditions. Results show that both 2A3 and 3H12 anti-sdAb mAbs bind to epitopes that seem sensitive to denaturation, and thus have a conformational epitope.
Table 3. Binding on native or denatured recombinant proteins by ELISA.
Characterization by ELISA on human Immunoglobulins
[00126] Selected anti-sdAb mAbs F233-2A3 and F233-3H12 were further characterized by ELISA on human immunoglobulins to assess their specificity to sdAbs only. To this end, ELISA was performed as described above with immobilized human lgG1 , lgG2, lgG3, lgG4, IgA, slgA and IgM along with positive control sdAb-1 ug13 and -1 ug36. Both 2A3 and 3H12 mAbs failed to bind to various human Ig antigens, except for positive controls 1 ug13 and 1 ug36 (Table 4). 3E6 is an anti-GFP negative control, and anti-hlgG (H+L) was used as a positive control for all human Ig.
Table 4. ELISA results of selected anti-sdAbs on purified human immunoglulins subclasses
EXAMPLE 4
EVALUATION OF AFFINITY OF ANTI-SDABS ON CAR-T CELLS mAbs purification
[00127] Selected mAbs supernatant were produced and purified on Protein G columns and desalted on HiPrep™ desalting columns pre-equilibrated in PBS and filter sterilized through 0.22 pM membrane (Millipore®). Purified mAbs were concentrated using Vivaspin™ turbo concentrators (30 kDa) and SEC purified on Superdex™ 200 to remove aggregates. The final concentration of the antibody solutions was determined using a Nano-drop™ 2000 (ThermoScientific®), using IgG as sample type. The recovery of mAb 3H12 was low due to its sensitivity to low pH required in protein G elution.
[00128] Sufficient quantity of the 2A3 mAb was purified and directly labelled with Alexa-Fluor™ 647 (AF-647) to assess its binding across a panel of llama sdAb-based CAR-T cells. The CAR-T constructs contained a P2A ribosomal self-skipping sequence separating the CAR from an EGFP marker, thereby allowing fluorescent detection of CAR-expressing cells. Thus, this GFP surrogate marker for CAR expression could be assessed against the AF-647 signal to assess the binding efficiency of 2A3 mAb.
[00129] Human T cells from 2 separate donors transduced with different sdAb-based CAR constructs were assessed for the level of CAR expression using a panel of anti-VHH antibodies by flow cytometry Results are depicted in Fig. 2.
[00130] Fig. 2 shows CAR expression detected by evaluating GFP expression versus binding of the anti- nH mAb 2A3. Un-transduced T cells (Mock) and CD19 scFv-based CAR were used as controls. [00131] Overall, the results indicate strong binding of the anti-VnH mAb 2A3 (APC/GFP signal > 0.5) to 11 /19 sdCAR expressing cells and weak for 8/19 with good correlation (R2=0.6) between CAR expression levels detected via GFP expression and binding of the anti-VnH mAb 2A3.
[00132] This assay was conducted using one set concentration (30nM) of 2A3. Increasing the 2A3 antibody concentration may be effective for increasing the binding to the weaker HH.
EXAMPLE 5
ANTIBODY SEQUENCING
[00133] The VH and V of each mAbs were sequenced by Sanger sequencing. Briefly, mRNA was extracted from hybridoma clones (Dynabeads mRNA Direct kit, ThermoFisher Scientific) and reverse transcribed into cDNA (Maxima H Minus First Strand cDNA with dsDNAse, ThermoFisher Scientific®). DNA encoding H and VL domains was PCR amplified (Q5 Hot Start High-Fidelity DNA Polymerase™, NEB™) using mixtures of degenerate forward primers annealing in FR1 and a single reverse primer annealing in CH1 (Novagen™ Mouse Ig Heavy, Kappa and Lambda Primer sets). The resulting amplicons were sequenced by regular Sanger sequencing using their respective forward and reverse primers. The DNA sequence of each H and VL domains were analyzed and then translated in silica. The CDRs sequence were determined using Kabat CDR numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi). The amino-acid sequences of the VH and VL are shown in Fig. 3. The sequence analysis revealed that clones 2A3 and 3H12 have unique VH and VL CDRs.
EXAMPLE 6
RECOMBINANT ANTIBODY PRODUCTION, PURIFICATION AND EVALUATION BY ELISA
[00134] To facilitate large scale antibody purification and consistency between productions, the anti-sdAbs monoclonal antibodies identified in Example 2 were produced recombinantly in CHOC3117 or CHO55E1 cells as stable pools.
[00135] The VH and V regions were cloned as fusions with mouse lgG2a/kappa constant regions (mouse lgG2a heavy chain and mouse kappa light chain, respectively) into the pTT109 vector.
[00136] The amino acid sequences of the recombinant antibodies: recF233-2A3-mlgG2a and recF233-3H12-mlgG2a is provided in the sequence table (SEQ ID NOs: 36, 37, 38 and 39, respectively). All light chain sequences comprise a signal sequence MRLPAQLLGLLMLWVSGSSG (SEQ ID NO:40) at the N-terminus, while heavy chain sequences comprised the signal sequence MPLLLLLPLLWAGALA (SEQ ID NO:41) at the N-terminus. [00137] Recombinant antibody expression was validated via a 2 mL expression scout. Briefly, CHO cells were transfected with VL and VH containing constructs (1 :1 ratio). Conditioned medium (CM) was harvested on day 7, recombinant antibodies were purified on Protein A (MabSelect SuRe), buffer exchanged on HiPrep™ desalting column, sterile-filtered, quantitated, and evaluated by UPLC-SEC and SDS-PAGE. The data showed that both recombinant antibodies were well expressed by the stably transfected CHO pools.
[00138] To confirm that the recombinantly expressed antibodies behave similarly to the hybridoma-expressed monoclonal antibodies, ELISA binding experiments on antigens sdAb-1 ug13, sdAb-1 ug36, FC5-MOD or human IgG (negative control) were performed as described in Example 1. All incubations were done at room temperature. Instead of hybridoma supernatant as primary antibody, serial 1/3 dilutions of purified mAbs starting at 50 nM in blocking buffer were added in duplicates. The data were analyzed with GraphPad Prism™ v 9.0 software using one-site specific binding with Hill slope non-linear regression curve fit model to determine Kdapp (concentration needed to achieve a halfmaximum binding at equilibrium) for each mAb tested.
[00139] All chimeric antibodies bind with similar specificity and similar or slightly better affinity to tested antigens (Table 5 and Fig. 4). The increased affinity obtained with the recombinant mAbs may be due to less stringent elution conditions used for protein A purification compared to original mAbs purified using protein G. All mAbs were negative on human IgG antigen (data not shown). Both recombinant mAbs have similar affinities to FC5-MOD or sdAb-1 ug13 antigen. RecF233-3H12 has a better affinity (5-fold) to sdAb-1 ug36 antigen compared to recF233-2A3.
Table 5: Apparent KD of anti-sdAb mAbs as determined by ELISA analysis.
[00140] The recombinant anti-VnH mAb was also tested for their ability to detect surface expression of sdAb-based CAR constructs as well as for their stability when stored over time at -80°C. To perform this assessment, serial dilution of the recombinant antibodies was added to 96-well plate followed by addition of respective CAR-T cells. The cells were allowed to incubate with the antibody for 15 minutes at room temperature. Cells were then centrifuged, washed and the secondary detection antibody; Goat F(ab')2 anti-mouse IgG (H+L) Alexa-Fluor™ 647 was added to the cells. Cells were further incubated for 15 minutes at room temperature, washed and then analyzed by flow cytometry. Fig. 5 depicts the binding of the anti-VnH mAb 2A3 and 3H12 to Jurkat T cells stably expressing sdCD22 CAR constructs. The binding curves for each antibody at time of production and at various time points post storage at -80°C are depicted. Results demonstrate strong and dose dependent binding of both 2A3 and 3H12 anti-VHH mAb to CD22 sdAb based CAR constructs expressed on Jurkat T cells. Results demonstrate that these antibodies show similar binding kinetics and thus are stable for at least 12 months when stored at -80°C.
EXAMPLE 7
ANTI-VHH ANTIBODIES SHOW DIVERGENT EPITOPE BINDING PROPERTIES AS ASSESSED VIA HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY
[00141] Recombinant anti-VHH antibody samples were produced as described in example 6 and utilized here. Purified sdAb-1 ug36 protein was equilibrated with a saturating concentration of each anti-VHH antibody at a 1 :1 molar ratio. Deuterium labelling of the antibody-VHH complexes or sdAb- 1 ug36 alone was initiated by a two-fold dilution with deuterated buffer (phosphate buffered saline in 90% D2O). A controlled time period of either 0.5 minutes or 3 minutes was allowed before the reaction was quenched by a 5-fold dilution with 1 M Guanidine HCI 40mM TCEP in 0.1% formic acid at 4°C. Samples were then trapped, washed, and digested with immobilized pepsin. Mass spectrometry was performed and deuteration was measured in triplicate for 41 peptides covering 81 % of the sdAb-1 ug36 sequence. Fig. 6 depicts the difference in the relative level of deuterium between the sdAb-1 ug36/anti- VHH complexes and unbound sdAb-1 ug36 observed via a mass shift in specific peptides within the analyzed sdAb-1 ug36 sequence. Highlighted values demark significant differences in the deuteration content in peptides between the sdAb-1 ug36/anti-VHH complexes and unbound sdAb-1 u36 based a pooled T-test (1-p = 0.98, 3x standard deviation cut-off). These results demonstrate a partially overlapping, but divergent binding mode for 2A3 and 3H12 anti-VHH antibodies. Specifically, 2A3 shows decreased deuteration in peptide region 2 upon binding whereas 3H12 shows decreased deuteration in peptide regions 1 , 2 and 3 upon binding, indicating that antibodies are likely interacting with different amino acid residues on the VHH proteins.
EXAMPLE 8
ANTI-VHH ANTIBODIES SHOW BROAD REACTIVITY WITH DIVERSE LLAMA VHH PROTEINS VIA ELISA
[00142] Recombinant anti-VHH antibody samples were produced as described in example 6 and utilized here. A total of 76 purified VHH proteins with diverse sequences and antigenic specificites were examined for reactivity with anti-VHH antibodies via ELISA. In brief, VHH protein was passively absorbed on a 96-well Immulon 4HBX plate in PBS buffer at 4°C overnight before blocking with PBS + 5% milk. Anti-VHH antibodies were then added to the plate at a known concentration and incubated at room temperature for 60 mins to allow antibody-antigen interaction. Plates were then washed 3 times with PBS + 0.05% Tween20 buffer. The amount of bound anti-VHH antibody was detected using anti-mouse HRP antibody, followed by washing. Finally, the TMB buffer was added for quantitation of HRP bound to the plate, the reaction was stopped with 1 M sodium phosphate, and assessed using a plate reader.
[00143] Fig. 7 depicts the amount of bound anti-VHH antibody detected for 2A3, 3H12, or a cocktail of both antibodies against 76 different VHH proteins. Results show shows 75 of 76 unique VHH’s are recognized by a cocktail 1 :1 mix of the anti-VHH antibodies; 74 of 76 are recognized by 3H12 mAb; and 73 of 76 are recognized by 2A3 mAb. Overall, these results demonstrate that 2A3 and 3H12 both show broad reactivity to many llama VHH proteins, with distinct binding patterns. A 1 :1 mix of both antibodies provides the best overall breadth of binding to diverse VHH proteins.
EXAMPLE 9
RECOMBINANT ANTIBODY LABELING AND SPECIFICITY ASSESSMENT
[00144] Antibody samples (2mg/mL) in PBS with 100 mM sodium carbonate pH 8.3 were incubated with Alexa Fluor™ 647 TFP-esterdye reagent (ThermoFisher Cat# A20173) at a dye:protein molar ratio of 10:1 for 1 hour at 25°C with mild agitation. The reactions were stopped, and removal of unincorporated excess dye was performed with 3 consecutive 10 mL Zeba™ (ThermoFisher cat# 89894) spin desalting columns pre-equilibrated with PBS pH 7.4. Purified samples were centrifuged for 10 minutes at 20,000 x g at 4°C, followed by quantification via absorption spectroscopy. The degree of labeling (DOL) and concentration were based on absorbance readings at 280 nm and 650 nm using the equation below. Monomeric purity was determined using analytical HPLC Size-exclusion chromatography on a Cytivia™ Superdex™ 200 Increase 5/150 GL column (Cytivia Cat# 28-9909-45) and PBS supplemented with 0.2 M arginine pH 7.2 as the running buffer.
[00145] To calculate the concentration of protein in the sample the following equation was used:
Protein concentration (m) = ([A28o-(Adye x CV280)] x dilution factor)/208000
[00146] Where 208,000 is the molar extinction coefficient (E) in cm-1M'1 of the variant prior to labeling. The value CF280 is 0.3, a correction factorforthe fluorophore’s contribution to the absorbance at 280 nm.
[00147] To calculate the degree of labeling the following equation was used:
Moles dye per mole protein = (Adye x dilution factor)/(Sdye x protein concentration (M) ) [00148] Where Edye (in cm-1M'1) is the approximate molar extinction coefficient of the Alexa Fluor™ 647 dye (239,000).
EXAMPLE 10
DETECTION OF SURFACE EXPRESSION OF SDAB-CAR BY RECOMBINANT ANTI-VHH ANTIBODY
[00149] Two different lots of AF-647 labeled recombinant anti-VnH mAb were tested for their ability to detect surface expression of sdAb-based CAR constructs. To perform this assessment, serial dilution of the recombinant antibodies was added to 96-well plates followed by addition of respective CAR constructs. The cells were allowed to incubate with the labelled anti-VnH antibody for 15 minutes at room temperature. Cells were then centrifuges, washed and analyzed for binding of the labelled anti-VnH antibody by flow cytometry. Results depicted in Fig. 7 demonstrate strong staining of Jurkat cells stably expressing CD22-sdAb-CAR constructs. Antibodies were also tested for stability at -80°C over 12 months using similar binding assay. Noting the shift in brightness of staining are due to changes in flow cytometer settings, whereas there was no significant change in apparent binding affinity for antibodies over the course of the 12 month test period. These results demonstrate that an AF-647 labelled antibody reagent can be stably stored for at least 12 months at -80°C.
[00150] Antibodies were also tested for use as a combined reagent in order to maximize the diversity of VHH proteins that can be identified with a labelling reagent. To test this, cells stably expressing EGFR or CD22-specific sdAb-CAR proteins were stained with AF-647 labelled 3H12 antibody, 2A3 antibody, ora mixture of both antibodies. Results depicted in Fig. 8 show higher apparent affinity for the mixed antibody reagent, demonstrating that a mixed antibody product can be employed to label sdAb proteins on the surface of cells. The use of the anti-VnH antibodies to detect the expression of sdAb in the context of other immunotherapy including but not limited to enumerating CAR expression on CAR modified NK cell therapy and to quantify titer of CAR expressing viral vectors has thus been demonstrated.
[00151] In order to confirm that binding of anti-VnH antibodies can be employed to specifically label cells that are expressing a sdAb-CAR protein, cells were stained with either AF-647-labelled anti- VnH (2A3) or a CD22-protein directly labelled with FITC. As shown in Fig. 9, both staining strategies reveal a similar proportion of CD22-specific CAR-expressing cells. These results demonstrate that anti- VnH antibody can be used to effectively enumerate the number of CAR expressing cells, with similar results to a directly fluorescently labelled recombinant target protein.
EXAMPLE 11 ASSESSMENT OF CELL SURFACE BINDING FOR SOLUBLE SDAB USING LABELLED ANTI- VHH ANTIBODY
[00152] In order to assess whether anti-VnH antibodies can be used as a secondary labelling reagent to test the cell binding characteristics of soluble sdAb proteins, the following experiments were performed. Firstly a cell staining experiment to test CD22-specific binding of soluble VHH proteins was performed as shown in the diagram in Fig. 8 left. In brief, high purity VHH proteins for CD22-specific antibodies (1 ug13 or 1 ug36) or a control single domain antibody specific for an irrelevant bacterial protein (B131) were produced using a standard bacterial expression system and metal affinity column purification. The Ramos CD22 expressing human lymphoma line, or a modified cell line wherein CRISPR technology has been employed to specifically disrupt CD22 expression, were combined with varying concentrations of these purified VHH proteins. Cells were then washed and stained with the mixed AF-647 labelled 2A3 and 3H12 anti-VnH reagent as a secondary antibody. Following staining, cells were again washed before examination via flow cytometry. As shown in Fig. 8, clear dosedependent binding to CD22+ cells could be observed for CD22+ target cells stained with 1 ug13 or 1 ug36 sdAbs (Fig. 8 centef) but not the negative control B131 antibody (Fig. 8 right). No binding was observed for CD22-deficient cells. These results demonstrate the anti-VnH antibodies can be effectively employed to examine cell binding of soluble sdAb molecules.
[00153] To assess whether a similar approach is broadly applicable for a wider variety of molecules, similar experiments were performed using purified BCMA-specific sdAb molecules. These molecules were then tested for binding to BCMA-high RPMI8226 cells, BCMA-low Raji cells, or BCMA- negative Jurkat cells. As shown in Fig. 11 , BCMA-sdAb proteins showed varying binding to BCMA- high and BCMA-low cells, and no apparent binding to BCMA-negative target cells. A similar experiment was performed to test a variety of mesothelin (MSLN)-specific sdAb proteins for binding to mesothelin- high H292 cells. As shown in Fig 12. MSLN-sdAb proteins displayed varying binding to cells. Overall, these results demonstrate that anti-VnH antibodies can be used to assess the relative binding of soluble sdAb proteins to various target cell line via flow cytometry.
EXAMPLE 12
ASSESSMENT OF TISSUE AND CELL LEVEL BINDING OF SOLUBLE SDAB USING LABELLED ANTI-VHH ANTIBODY IN IMMUNOHISTOCHEMISTRY
[00154] In order to demonstrate that these anti-VnH reagents can be used for assessment of tissue and cell specificity of a soluble sdAb reagent, a workflow as shown on Fig. 13 left was developed wherein tissue samples are stained with a purified sdAb, probed with anti-VnH secondary antibody, and finally detected using a commercial anti-mouse antibody staining reagent. In the data shown here, a frozen tissue array with various human tissues was obtained and stained with a purified CD22- specific sdAb or a control antibody. These samples were then washed and stained with anti-VnH secondary antibody. Finally, slides were washed and stained with an anti-mouse detection reagent. As shown in Fig. 13 right, clear staining was observed for tissues expected to have CD22+ cells such as spleen, lymph node, and bone marrow but no strong specific staining could be observed in nonlymphoid tissues such as brain, skin, and kidney. Overall these results demonstrate that anti-VnH antibodies can be employed to assess tissue and cell level binding of purified sdAbs in histology samples.
EXAMPLE 13
ACTIVATION AND EXPANSION OF SDAB-CAR BY LABELLED ANTI-VHH ANTIBODY
[00155] In addition to use of the anti-VHH for detection and enumeration of sdAb based CAR constructs and viral vectors, or binding of soluble sdAb proteins, the ability of these anti-VnH antibodies to directly stimulate activation of sdAb-CAR-T cells when immobilized on a solid surface such as adsorbed onto a microtiter plate has also been demonstrated. Such direct CAR activation could be used as a method of specifically expanding CAR-expressing immune cells, or as a possible potency assay for sdAb-based CAR constructs to be used as a release assay for CAR products. An additional application might be in the use of a recombinant bi-specific antibody technology to specifically redirect CAR-T responses analogous to BITE therapeutics.
[00156] To demonstrate the ability of the anti-VnH mAbs to activate CAR transduced T cells; 96-well flat-bottomed microtiter plates were coated with varying concentrations of anti-VHH mAb (3H12 in the examples depicted herein) or the anti-CD3 mAb OKT3 (positive control). CAR-T cells (10K/well) were added to each well and plates incubated for 24 hrs at 37°C in a CO2 incubator and assessed for activation by flow cytometry at the end of incubation time. The expression of early T cell activation marker CD69 was used as a surrogate measure for T cell activation.
[00157] The native antigen-specific T-cell receptor molecules on human T cells are non- covalently associated on the cell surface with the CD3 molecular complex. Immobilization of this complex with surface-bound anti-CD3 monoclonal antibodies induces T cell activation without the need for antigen-specific recognition via the T cell receptor. Therefore, as expected, dose dependent activation of all T cells including un-transduced mock T cells and both sdAb and scFv-based CAR transduced T cells were observed in response to OKT3; the well-established T-cell activating anti-CD3 mAb which was used as the positive control (Fig. 14A). In contrast to CD3 targeting, the anti-VnH reagent used here specifically targets the sdAb domain of sdAb-CAR cells when immobilized on a 2D surface. Furthermore anti-VnH should not activate non-transduced T cells (mock) or single-chain variable fragment CAR-expressing cells (scFv-CAR). As expected, a dose dependent activation was only observed in sdAb-based CAR constructs with the anti-VnH mAb. No activation was seen in response to anti-VnH mAbs in untransduced T cells or scFv-based CAR-T cells (Fig. 14B).
[00158] To demonstrate the ability of surface immobilized anti-VnH mAbs to expand CAR transduced T cells; 96-well flat-bottomed microtiter plates were coated with 50 pg/mL concentration of anti-VnH mAb 3H12. CAR-T cells (10K/well) were added to each well and plates were placed in a Sartorius IncuCyte® S3 (Essen Bioscience™) device and incubated for 5 days at 37°C. On day 6 and every 3 days thereafter, cells were re-suspended by gentle mixing and 1/5th volume of the cell suspension was transferred to a new plate containing fresh media without anti-VnH mAb. Phase confluence of the cells was monitored over time using IncuCyte® analysis software. Fig. 15 depicts the measure of cell expansion over time. Strong expansion of all 4 sdAb-based CAR-T cells were observed over time up to at least day 15 post exposure to the anti-VnH mAb. No such expansion was observed with the scFv-based CAR-T or un-transduced mock T cells suggesting anti-VnH mAb were capable of selectively expanding sdAb-based CAR constructs.
EXAMPLE 14
THE APPLICATION OF ANTI-VHH ANTIBODIES TO INHIBIT SDAB-CAR FUNCTION
[00159] In addition to use of the anti-VnH for detection and enumeration of sdAb based CAR constructs and viral vectors, or binding of soluble sdAb proteins, the ability of soluble anti-VHH antibodies to bind to VHH CARs can be used to inhibit the functional effect of a VHH-CAR expressing cell. Fig 16 depicts the hypothetical mechanism by which an anti-VHH antibody might bind to a VHH and interrupt the cellular mechanisms required for activation of the CAR-expressing cells.
[00160] Fig. 17 depicts the results of a cell activation experiment demonstrating direct inhibition of an EGFR-targeted VHH-CAR by varying concentrations of anti-VHH antibodies alone or in combination. The immortalized human T cell line, Jurkat, was used for this experiment. These cells were engineered using lentiviral gene transfer to specifically express a VHH-CAR targeted towards the human EGFR receptor (as described in McComb et al, Front. Immunol., 21 July 2022, Vol. 13 (https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.864868). EGFR-CAR cells were then placed at 37°C for 48 hours in co-culture with EGFR-positive SKOV3 human lung cancer cells, in the presence of decreasing concentrations of the anti-VHH antibodies, 2A3 and 3H12, alone or in combination, or vehicle control. Whereas EGFR-CAR cells in the absence of target cells show low expression of the CD69 activation marker (open squares), the addition of EGFR-positive target cell lines results in a strong upregulation of CD69 (open diamonds). With increasing concentration of the anti-VHH antibodies alone or in combination we see a dose-dependent inhibition of the activation of EGFR-VHH-CAR cells in response to target cells. These results demonstrate that soluble VHH antibodies are able to directly inhibit the activation of VHH CAR cells.
EXAMPLE 15
DIRECT KILLING OF SDAB-CAR EXPRESSING CELL USING ANTI-VHH ANTIBODY-DRUG CONJUGATE
[00161] Using the broadly-reactive anti-VnH antibody of the present invention, it is also possible to directly kill cells which express the VHH target molecule(s) on their surface. This may be employed as a safety reagent to deplete VHH-CAR expressing cells in the event of pathogenic growth of therapeutic cells in a human patient. To demonstrate this effect, DM1 cytotoxic drug is conjugated to purified recombinant F233-3H12 antibody using an in house drug conjugation method. In brief, antibody was conjugated to the microtube inhibitor mertansine (DM1) via the bifunctional cross-linker Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) using a 1-step synthesis method where N2'-Deacetyl-N2'-[3-[[1-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]- 2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]-maytansine (SMCC-DM1) with an active N- hydroxysuccinamide ester functional group was randomly reacted with an antibody surface accessible lysine primary amine group. SMCC-DM1 solubilized in dimethyl acetamide was mixed with antibodies in 100 mM Sodium phosphate, 20 mM NaCI, 2mM EDTA pH 7.4, with a final DMA co-solvent concentration of 5% v/v and a SMCC-DM1 :antibody molar ratio of between 7 and 12 to 1. This mixture was incubated at room temperature overnight with no agitation. Conjugated antibody was purified using desalting columns into formulation buffer consisting of 20 mM Sodium succinate, 0.02% Polysorbate- 20 pH 6.0. Anti-VHH-DM1 antibody drug conjugate (ADC) was then purified using affinity column. This DM1 conjugated anti-VHH antibody is referred to as 3H12-DM1 below.
[00162] A cytotoxicity assay is then performed using 3H12-DM1 two human Jurkat T cell line, one without CAR expression (WT) and one with VHH-CAR expression. Varying concentrations of the 3H12-DM1 were mixed with the Jurkat cells and incubated the cells overnight. The next day, viability is assessed using flow cytometric assessment of cell condition. Fig. 18 shows a sharp drop in Jurkat cell viability for those cells that express the VHH-CAR, but no effect on WT Jurkat cells. These results demonstrate that Anti-VnH antibodies can be used effectively to specifically kill VHH-CAR expressing cells.
EXAMPLE 16
REDIRECTION OF CAR-EXPRESSING CELLS TOWARDS ADDITIONAL PROTEIN TARGETS
[00163] The anti-VnH reagent of the present invention may also be used to redirect the cytotoxicity or other cellular responses mediated by a VHH containing protein using a soluble VHH- redirector protein. In this case, an scFv or other antibody derivative molecule would be linked to an additional antigen binding element, which could be scFv, sdAb, or another antigen targeting moiety. This would then effectively redirect responses against alternative targets. This could be used for targeting multiple antigens on a single cell, for example, in the event that the first antigen targeted by the VHH containing protein would be downregulated or targeting alternative cells altogether. Fig. 19 shows a diagram of this application.
[00164] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.
SEQUENCES

Claims

CLAIMS:
1. A composition comprising an anti-VHH antibody, comprising an antibody or antigen-binding fragment thereof, that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), and three variable light domain CDR (CDR L1 , L2 and L3), wherein composition comprises: a) a first anti-VHH antibody, said CDR H1 , H2, H3, L1 , L2, and L3 comprising the amino acid sequences:
CDR H1 : GYGIS (SEQ ID NO:1),
CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
CDR H3: FYFELAY (SEQ ID NO:3),
CDR L1 : RTNLGGNYMY (SEQ ID NO:4),
CDR L2: YTSNLAP (SEQ ID NO:5), and
CDR L3: QQFTSSASTWT (SEQ ID NO:6), respectively, in admixture with a diluent, excipient, or carrier; or b) a second anti-VHH antibody, said CDR H1 , H2, H3, L1 , L2, and L3 comprising the amino acid sequences:
CDR H1 : NYHMS (SEQ ID NO:7),
CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8),
CDR H3: QRSDYWFDY (SEQ ID NO:9),
CDR L1 : RASENIYSYLA (SEQ ID NO:10),
CDR L2: NAKTLTE (SEQ ID NO:11), and
CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively, in admixture with a diluent, excipient, or carrier; or c) both the first and the second anti-VHH antibody.
2. The composition of claim 1 , wherein the first anti-VHH antibody comprises
• a variable heavy domain (VH) comprising the amino acid sequence:
QAQLQQSGAELARPGASVRLSCKASGYTFTGYGISWVKQRTGQGLEWIGEIYPGSGSIYYNEKFKG TATLTADKSSSTAYMQLSSLTSEDSAVYFCARFYFELAYWGQGTLVTVSA (SEQ ID NO:29), and
• a variable light domain (V ) comprising the amino acid sequence: EIVLTQFPAIISASLGEKVTMNCRTNLGGNYMYWYQQKSDASPRLWIYYTSNLAPGVPARFSGSGSG
NSYSLTISSMEGEDAATYYCQQFTSSASTWTFGGGTKLEIK (SEQ ID N0:31). and the second anti-VHH antibody comprises
• a variable heavy domain (VH) comprising the amino acid sequence:
EVQLVESGGGLVQPGGSLKLSCAASGFSFSNYHMSWFRQTPEKRLEWVAYISSGGGSTFYPDSVK GRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRSDYWFDYWGQGTTLTVSS (SEQ ID NO:30), and
• a variable light domain (VL) comprising the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWFQQRQGKSPHLLVYNAKTLTEGVPSRFSGSGS GTQFSLKINSLQPEDFGSYYCQHHYGTLFTFGSGTKLEIK (SEQ ID NO:32).
3. The composition of claim 1 or 2, wherein said anti-VHH antibody is humanized or partially humanized.
4. The composition of any one of claims 1 to 3, wherein said anti-VHH antibody is bound directly or through a linker to a functional molecule.
5. The composition of claim 4, wherein the functional molecule is a detectable label, a peptide, a protein, an enzyme, a nucleic acid, a lipid particle, a cytotoxic agent, a drug, a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof.
6. The composition of claim 5, wherein said detectable label is a fluorescent marker, a radioactive marker, an MRI contrast agent, a detectable secondary antibody, or a combination thereof.
7. The composition of claim 4, 5, or 6, wherein said anti-VHH antibody is bound to the functional molecule via a peptide linker comprising 1 to 40 amino acid residues.
8. The composition of claim 7, wherein the peptide linker comprises the amino acid sequence (SS)n, (GGG)n, (GGGG)n, (GGGS)n, (SSGGG)n, (SEQ ID NO: 33), or (GGGGS)n (SEQ ID NO: 34), wherein n > 1 .
9. A composition comprising an anti-VHH antibody, consisting of a light chain only or a heavy chain only antigen-binding antibody fragment, that binds specifically to a Camelid variable heavy domain of heavy chain (VHH), wherein the anti-VHH antibody comprises an amino acid sequence comprising:
• three variable heavy domain complementarity determining regions (CDR)(CDR H1 , H2 and H3), consisting of either:
CDR H1 : GYGIS (SEQ ID NO:1),
CDR H2: EIYPGSGSIYYNEKFKG (SEQ ID NO:2),
CDR H3: FYFELAY (SEQ ID NO:3), or or
CDR H1 : NYHMS (SEQ ID NO:7),
CDR H2: YISSGGGSTFYPDSVKG (SEQ ID NO:8),
CDR H3: QRSDYWFDY (SEQ ID NO:9); or
• three variable light domain CDR (CDR L1 , L2 and L3), consisting of either:
CDR L1 : RTNLGGNYMY (SEQ ID NO:4),
CDR L2: YTSNLAP (SEQ ID NO:5), and
CDR L3: QQFTSSASTWT (SEQ ID NO:6), respectively; or
CDR L1 : RASENIYSYLA (SEQ ID NQ:10),
CDR L2: NAKTLTE (SEQ ID NO:11), and
CDR L3: QHHYGTLFT (SEQ ID NO:12), respectively; in admixture with a diluent, carrier, or excipient.
10. The composition of any one of claims 1 to 9, wherein the anti-VHH antibody is operable
• to either stimulate or block the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell having a VHH on the surface;
• to detect a VHH on the surface a cell;
• to redirect a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR with a surface VHH; or
• to assess the binding properties of a VHH.
11. A nucleic acid molecule encoding the anti-VHH antibody as defined in any one of claims 1 to 9, a vector comprising said nucleic acid molecule operably linked to one or more regulatory elements to allow expression of the anti-VHH antibody as defined in any one of claims 1 to 9 in a host cell, or a cell comprising said vector for expressing the anti-VHH antibody as defined in any one of claims 1 to 9.
12. A method forthe detection of a VHH on the surface a cell comprising detecting said VHH using the composition of any one of claims 1 to 9.
13. A method for either stimulating or blocking the activation, the expansion, or both the activation and the expansion of a chimeric antigen receptor (CAR)-T cell having a VHH on the surface, comprising contacting said CAR-T cell with the composition of any one of claims 1 to 9.
14. A method for redirection of a response of a chimeric antigen receptor (CAR)-T cell comprising a CAR with a surface VHH, comprising contacting said CAR-T cell with the composition of any one of claims 1 to 9, wherein the anti-VHH antibody comprises a second antibody, an antibody fragment, a second antigen-binding fragment, or a combination of any two or more thereof, specific for a second antigen different from the antigen for which the CAR is specific.
15. A method for assessing the binding properties of a VHH, comprising subjecting a sample in vivo, ex vivo, or in vitro to purified VHH and then detecting binding of said VHH using the composition of any one of claims 1 to 9.
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