EA044007B1 - ANTIGEN-BINDING REGIONS AGAINST TYPE III FIBRONECTIN DOMAINS AND METHODS OF THEIR APPLICATION - Google Patents

Description

The currently pending application contains a sequence listing provided electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the above list in ASCII format, created on June 29, 2018, is called

JBI5032WOPCTSL.txt and is 88120 bytes in size.

Field of technology

This invention relates to antibodies that specifically bind to fibronectin type III domains (FN3 domains), as well as methods for producing and using the described antibodies.

Prerequisites for creating the invention

Fibronectin scaffolds are a family of proteins that can be modified to bind to any compound of interest. These proteins, which typically use a scaffold derived from a fibronectin type III (FN3) or FN3-like domain, function in a manner characteristic of natural or engineered antibodies (i.e., polyclonal, monoclonal, or single-chain antibodies), and, in addition, Moreover, they have structural advantages. In particular, the structure of such antibody mimics has been designed for optimal folding, stability and solubility, even under conditions that would normally result in loss of antibody structure and function. An example of fibronectin-based scaffold proteins is Centyrin™ (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; US 2010/0216708).

Fibronectin type III (Fn3) domains contain N-terminal to C-terminal beta or beta-like strand A; loop AB; beta or beta-like strand B; BC loop; beta or beta-like strand C; CD loop; beta or beta-like strand D; loop DE; beta or beta-like strand E; loop EF; beta or beta-like strand F; loop FG; and beta or beta-like strand G. Any or all of loops AB, BC, CD, DE, EF and FG may participate in target binding. The BC, DE and FG loops are structurally and functionally similar to the complementarity determining regions (CDRs) of immunoglobulins.

Given their small size, lack of disulfide bonds, high stability, and ability to be expressed in prokaryotic hosts, FN3 domains have been of biopharmaceutical interest. FN3 domains can be readily conjugated to drugs/toxins, efficiently penetrate tissues, and can be readily formulated into multispecific binders and fusion proteins, including chimeric antigen receptors (CARs).

Despite its versatility, there are currently no antibodies available that specifically bind to FN3 domains for detection, analysis, or biopharmaceutical purposes.

Brief description of the invention

This invention includes antibodies and antigen-binding fragments that bind to a non-randomized region of the fibronectin type III domain (FN3). Also described are related polynucleotides capable of encoding the proposed antibodies to the FN3 domain and antigen-binding fragments, cells expressing the proposed antibodies and antigen-binding fragments, as well as corresponding vectors and detectably labeled antibodies to the FN3 domain and antigen-binding fragments. The antibody or its antigen-binding fragment does not selectively bind to the randomized region of the FN3 domain as determined by ELISA under the conditions described in Example 3.

Moreover, methods of using the proposed FN3 domain antibodies and antigen binding fragments are described. The described anti-FN3 domain antibodies and antigen binding fragments can be used to detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells. In another embodiment, the disclosed anti-FN3 domain antibodies and antigen binding fragments can be used to activate T cells expressing CARs containing FN3 domains. In yet another embodiment, the described anti-FN3 domain antibodies or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the invention includes isolated antibodies and antigen binding fragments, wherein the antibody or antigen binding fragment specifically binds to a non-randomized region of the FN3 domain. Expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells can be detected using these FN3 domain antibodies or antigen binding fragments thereof. In some embodiments, anti-FN3 domain antibodies or antigen-binding fragments activate T cells expressing CARs containing FN3 domains. In some embodiments, anti-FN3 domain antibodies and antigen binding fragments bind to an FN3 domain that is modified in the loop regions. In table 1 shows the CDR sequences of the FN3 domain-specific antibody described herein.

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Table 1

CDR sequences of antibodies specific to the FN3 domain (SEQ ID NO:)

Defining boundaries HCCDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 IMGT GIDLSTS V (1) IYTNVNT (4) ARAVYAGA MDL (7) ERIYSN (9) KAS (11) QYTSYGS GYVGT (13) Kabat TSVMG (2) FIYTNVNTYY ASWAKG (5) AVYAGAMD L(8) QASERIYSN LA (10) KASTLAS (12) QYTSYGS GYVGT (13) Chotiya GIDLSTS (3) YTNVN (6) AVYAGAMD L(8) QASERIYSN LA (10) KASTLAS (12) QYTSYGS GYVGT (13)

Defining boundaries HCCDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 IMGT GFSLNT SGTG (35) IWWDDDK (41) VRIKGRMDY (44) QSVLFGSK QKNY (46) WAS (48) HQYLSLF T (50) Kabat TSGTGV G(36) HIWWDDDKG YNPALKS (42) IKGRMDY (45) KSSQSVLFG SKQKNYLA (47) WASTRES (49) HQYLSLF T (50) Chotiya GFSLNT SGT (37) WWDDD (43) IKGRMDY (45) KSSQSVLFG SKQKNYLA (47) WASTRES (49) HQYLSLF T (50)

Defining boundaries HCCDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 IMGT GIDFSS VAY (38) IYAGSSSSI (51) ARGLFTSGS GYYIDM (54) QSIGSD (56) SAS (58) QCTYSSST GYNA (60) Kabat SVAYM CIYAGSSSSSIY GLFTSGSGY QASQSIGSN GASNLAA QRGYISSA C (39) YASWAKG (52) YIDM (55) LA (57) (59) VDFFV (61) Chotiya GIDFSS YAGSSSS (53) GLFTSGSGY QASQSIGSN GASNLAA QRGYISSA VA (40) YIDM (55) LA (57) (59) VDFFV (61)

In some embodiments, the anti-FN3 antibody or antigen-binding fragment thereof comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences described in Table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any of the amino acid sequences described in table. 1. The anti-FN3 domain antibodies of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 14 and may comprise a light chain variable region sequence of SEQ ID NO: 15. In other embodiments, the anti-FN3 domain antibodies of the invention may comprise a heavy chain variable region sequence SEQ ID NO: 74 and may comprise the light chain variable region of SEQ ID NO: 75. In other embodiments, the anti-FN3 domain antibodies of the invention may comprise the heavy chain variable region sequence of SEQ ID NO: 78 and may comprise the light chain variable region of SEQ ID NO: : 79.

Anti-FN3 domain antibodies described herein include antibodies with the described CDR and variable domain properties in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to affect various effector functions.

In addition to the described antibodies to the FN3 domain and antigen-binding fragments, polynucleotide sequences capable of encoding antibodies to the FN3 domain and antigen-binding fragments are also proposed. Also provided are vectors containing the described polynucleotides, as well as cells expressing antibodies to the FN3 domain or antigen binding fragments provided herein. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf9 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). A method for producing antibodies to the FN3 domain or antigen-binding fragments is also proposed.

The invention also includes a CAR of the invention comprising an isolated polypeptide comprising:

(a) an extracellular domain having an scFv that specifically binds to a non-randomized

- 2 044007 region of the FN3 domain;

(b) transmembrane domain; and (c) an intracellular signaling domain.

The CAR may further comprise a hinge region connecting the extracellular domain and the transmembrane domain.

In some embodiments, the CAR-derived polypeptide comprises:

(a) an extracellular domain comprising an FN3 domain of the invention, such as an FN3 domain having an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 68-73, preferably one of SEQ ID NOs: 68-73;

(b) a hinge region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, preferably SEQ ID NO: 24;

(c) a transmembrane domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, preferably SEQ ID NO: 25; and (d) an intracellular signaling domain comprising a co-stimulatory domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, preferably SEQ ID NO: 26, and a primary signaling domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27, preferably SEQ ID NO: 27.

The invention also includes an isolated polynucleotide encoding a CAR of the invention, comprising:

(a) an extracellular domain having an scFv that specifically binds to a non-randomized region of the FN3 domain;

(b) transmembrane domain; and (c) an intracellular signaling domain.

The CAR may further comprise a hinge region connecting the extracellular domain and the transmembrane domain.

In some embodiments, the isolated CAR-encoding polynucleotide comprises:

(a) an extracellular domain comprising an FN3 domain of the invention, such as an FN3 domain having an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 68-73, preferably one of SEQ ID NOs: 68-73;

(b) a hinge region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, preferably SEQ ID NO: 24;

(c) a transmembrane domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, preferably SEQ ID NO: 25; and (d) an intracellular signaling domain comprising a co-stimulatory domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, preferably SEQ ID NO: 26, and a primary signaling domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27, preferably SEQ ID NO: 27.

In yet another general aspect, the present invention relates to a CAR of the invention, a vector containing a polynucleotide encoding a CAR of the invention, and a host cell containing a vector or isolated polynucleotide encoding a CAR of the invention. The invention also relates to a method for producing a CAR according to the invention, comprising culturing a host cell containing a polynucleotide sequence encoding a CAR under CAR production conditions, and isolating the CAR. The CAR can be associated with a host cell or isolated cell membrane from a host cell.

In yet another general aspect, the invention relates to engineered immune cells containing the CARs of the invention. Preferably, the engineered immune cells are T cell receptor knockout immune cells. Preferably, the engineered immune cells are HLA-I/beta-2 microglobulin knockout immune cells. Optionally, HLA-I/beta-2-microglobulin knockout immune cells are additionally HLA-II knockout immune cells that lack allogeneic immune responses from the host patient. The engineered immune cells may contain a second CAR having an extracellular domain that specifically binds to a target other than the FN3 domain. The engineered immune cells may also be resistant to at least one anti-cancer chemotherapy.

In yet another general aspect, the invention relates to pharmaceutical compositions containing engineered immune cells according to the invention.

In yet another general aspect, the invention relates to a method of treating a B cell-related condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention. In a preferred embodiment, the B cell disease condition is multiple myeloma.

In yet another general aspect, the invention relates to a method for constructing an immune cell

- 3 044007 according to the invention, including providing an immune cell and introducing into the cell a polypeptide encoding a CAR according to the invention.

In yet another general aspect, the invention relates to a method for preparing a pharmaceutical composition, comprising combining an engineered immune cell according to the invention with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

The present invention provides kits, including disclosed antibodies to the FN3 domain or antigen binding fragments thereof. The described kits can be used to implement methods of using antibodies to the FN3 domain or antigen binding fragments proposed herein, or other methods known to those skilled in the art. In some embodiments, the described kits may contain anti-FN3 domain antibodies or antigen binding fragments described herein and reagents for use in detecting the presence of FN3 domains in a biological sample and, optionally, a vessel for storing the anti-FN3 domain antibody or fragment when not present. used, instructions for use of the anti-FN3 domain antibody or fragment, the anti-FN3 domain antibody or fragment attached to a solid support, and/or labeled to detect the form of the anti-FN3 domain antibody or fragment.

Brief description of the figures

In fig. 1A-C show binding testing of recombinant AS7B90 and AS7B91 to immobilized FN3 domains or negative control proteins and comparing each with the parent rabbit hybridoma-derived antibody CEN25-105-5. In fig. 1A shows binding data for tencon25, which has no target specificity. In fig. 1B shows binding data for A3, which is the human cMET-specific FN3 domain. In fig. 1C shows binding data for 83v2-ABD, which is a human EGFR-specific FN3 domain with an albumin-binding domain.

In fig. Figures 2A-M show that AS7B91 was able to detect all CARTyrins (centyrins in CAR format) expressed on the surface of T cells.

In fig. Figure 3 shows detection of AS7B91 scFv CAR expression on the surface of primary T cells using tagged tencon 25.

In fig. 4A-C show FN3 domain-specific AS7B91 scFv CAR-BCMA degranulation in response to target cells expressing BCMA. In fig. 4A shows H929 cells (cells with high expression of BCMA). In fig. 4B shows ARH77 (BCMA low expression cells). In fig. Figure 4C shows K562 (BCMA-negative cells). Various FN3 domains were incubated at 50 nM with AS7B91 scFv CAR T cells, washed and then added to target cells in a 1:1 ratio. Option 1 BAR-T=AS7B91 scFv L-H CAR; Option 2 BAR-T= AS7B91 scFv H-L CAR.

In fig. 5 shows AS7B91 scFv CAR-BCMA killing of target cells expressing BCMA. Cells with high expression of BCMA, H929; Cells with low expression of BCMA, ARH77; BCMA-negative cells, K562. Various FN3 domains were incubated at a concentration of 25 nM with AS7B91 scFv CAR T cells, washed and then added to the target cells in a 1:1 ratio. Option 1 BAR-T=AS7B91 scFv L-H CAR; Option 2 BAR-T=AS7B91 scFv H-L CAR.

In fig. 6 shows the binding of tagged Tencon-25 to AS7B16 scFv CAR constructs on T cells. L2H, light chain-heavy chain orientation AS7B16. H2L, heavy chain-light chain orientation AS7B16.

In fig. 7 shows binding of EGFR tagged 83v2 to AS7B16 scFv CAR constructs on T cells. L2H, light chain-heavy chain orientation AS7B16. H2L, heavy chain-light chain orientation AS7B16.

In fig. 8 shows the binding of tagged Tencon-25 to AS7B82 scFv CAR constructs on T cells. L2H, light chain-heavy chain orientation AS7B82. H2L, heavy chain-light chain orientation AS7B16.

In fig. 9 shows the binding of EGFR tagged 83v2 to AS7B82 scFv CAR constructs on T Cells. L2H, light chain-heavy chain orientation AS7B16. H2L, heavy chain-light chain orientation AS7B16.

In fig. 10 shows the generation of FcgR-expressing cell lines. Purified lentiviral expression plasmids encoding human FcgRs (CD16a, CD32, and CD64) were packaged for transfection of 293T cells using the Lenti-Pac HIV Expression Packaging System.

Fig. 11A, B. Conjugation of cyclic citrullinated peptides to centyrin Tepsop25 was carried out in a 1:5 ratio (centyrin to peptide) via sortase chemistry. The conjugates were manually purified on a Ni-Sepharose (GE) column to remove free sortase and peptide. After purification, the buffer was replaced with PBS and the conjugates were concentrated. Conjugates were quality checked by (a) mass spectrometry (LC-MS) and (b) size exclusion chromatography (Superdex 75) and subjected to sterile filtration.

Fig. 12. Detection of binding of anticitrullinated antibodies to centirinCCP1-peptide conjugates. HEK293 cells expressing FcgR were incubated with 200 μg/ml human anti-citrullinated fibrinogen antibody or human IgG1 isotype control. Binding was assessed using flow cytometry.

Fig. 13. AS7B91 scFv CAR T cell-mediated killing of cells expressing

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FcR bound to anticitrullinated mAb.

Fig. 14A-C. Evaluation of potential peptide/antibody combinations in multiple autoimmune diseases to expand the BAR-T platform. Peptides specific for a) myasthenia gravis (MG), b) multiple sclerosis (MS) and c) systemic lupus erythematosus (SLE) were tested for their ability to bind to anti-AChR, anti-MOG or anti-dsDNA antibodies, respectively. In fig. 14A disclose SEQ ID NOs 85-87, respectively, in order. In fig. 14B disclose SEQ ID NOs 88-90, respectively, in order. In fig. 14C disclose SEQ ID NOs 91-93, respectively, in order.

Detailed Description of the Invention

Definitions

In the Background to the Invention section and in the text of this application, citations or descriptions of various publications, articles and patents are provided; each of these references being incorporated herein by reference in their entirety. The discussion of documents, acts, materials, devices, products, and the like that have been included in the present description is provided as context for the present invention. Such discussion is not an admission that any or all such sources are part of the prior state of knowledge with respect to any inventions described or claimed.

When used in this specification and in the accompanying claims, the singular number includes the plural unless the content of the text clearly indicates otherwise. Thus, for example, a reference to a cell includes a combination of two or more cells and the like.

As used herein, the term about, when indicating a measurable quantity such as quantity, duration in time, etc., is considered to cover deviations of up to ±10% from the specified value, as such deviations are acceptable for implementation of the described methods. Unless otherwise indicated, all numbers expressing amounts of ingredients, characteristics such as molecular weight, reaction conditions, etc., used in the description and claims are to be understood as modified in all cases by the term about. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following description and the appended claims are approximate values that may vary depending on the desired properties that are desired to be obtained by the present invention. As a last resort, and not as an attempt to limit the application of the theory of equivalents to the scope of the claims, each numerical parameter should at least be considered in light of the number of significant figures presented and using standard rounding techniques.

Although the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values indicated in the specific examples are presented as accurately as possible. However, any numerical value by its nature contains certain errors, inevitably resulting from the standard deviation found in the corresponding test measurements.

The term isolated means that a biological component (e.g., nucleic acid, peptide, or protein) has been substantially separated from, derived separately from, or purified from other biological components of the organism in which the component occurs naturally, i.e., other chromosomal and extrachromosomal DNA , RNA and proteins. Thus, the nucleic acids, peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides and proteins can be part of a composition and still be considered isolated if such composition is not part of the original environment of the nucleic acid, peptide or protein. The term also includes nucleic acids, peptides and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, the term isolated antibody or antigen binding fragment means an antibody or antigen binding fragment that is substantially free of other antibodies or antigen binding fragments having different antigen specificities (e.g., an isolated antibody that specifically binds to the FN3 domain is substantially free of antibodies , which are not specific to FN3 domains).

As used herein, the term fibronectin type III domain or FN3 domain refers to a domain commonly found in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, PNAS USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:1565915665, 1990), or a derivative thereof. Examples of FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and synthetic non-naturally occurring FN3 domains, such as in US8278419. Individual FN3 domains are designated by domain number and protein name, for example tenascin FN3 domain 3 (TN3) or fibronectin FN3 domain 10 (FN10).

Antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD and IgY), including various monomeric, polymeric and chimeric forms, unless otherwise specified. In particular, the term antibody covers polyclonal antibodies, monoclonal antibodies (mAbs) and antibodies.

- 5 044007 similar polypeptides, such as chimeric antibodies and humanized antibodies.

Antigen-binding moieties are any protein structure capable of exhibiting binding affinity for a specific antigen. Antigen-binding fragments include fragments obtained by any known method, such as enzymatic digestion, peptide synthesis and recombinant methods. Some antigen-binding fragments are composed of portions of intact antibodies that retain the antigen-binding specificity of the original antibody molecule. For example, antigen binding fragments may contain at least one variable region (heavy chain or light chain variable region) or one or more antibody CDR regions known to bind to a particular antigen. Examples of suitable antigen binding fragments include, but are not limited to, diabodies and single chain molecules, as well as Fab, F(ab') ₂ , Fc, Fabc and Fv molecules, single chain (Sc) antibodies, single antibody light chains, single antibody heavy chains, chimeric fusions of antibody chains or CDRs with other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, monovalent fragment consisting of VL, VH, CL and CH1 domains, or a monovalent antibody described in WO2007059782, divalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region, an Fd fragment consisting essentially of V.sub.H and C.sub.H1 domains; An Fv fragment consisting essentially of the VL and VH domains of one arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and is also called a domain antibody (Holt et al;Trends Biotechnol.2003 Nov;21(11):484-90); camelid antibody or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24); selected complementarity determining regions (CDRs), etc. All antibody isotypes can be used to obtain antigen-binding fragments. In addition, antigen binding fragments may include non-antibody protein scaffolds into which polypeptide segments can be successfully inserted in an orientation conferring affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments can be produced recombinantly or by enzymatic or chemical digestion of intact antibodies. The phrase antibody or antigen binding fragment thereof may be used to indicate that the antigen binding fragment comprises one or more amino acid segments of an antibody referred to in the phrase.

The terms CDR and the plural of CDR refer to the complementarity determining region (CDR), with three such regions defining the binding nature of the light chain variable region (CDRL1, CDRL2 and CDRL3), and three defining the binding nature of the heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that include backbone or framework regions. The precise definition of the boundaries and extent of the CDR depends on various classification and numbering systems. Therefore, CDRs can be distinguished by numbering by IMGT, Kabat, Chotia or any other boundary definitions. Despite their different boundaries, each of these systems is characterized by a certain degree of overlap in that part that constitutes the so-called hypervariable region within the variable sequences. Thus, CDR definitions under such systems may differ in length and boundary regions relative to the adjacent frame region. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901 (1987); and MacCallum et al., Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography, J. Mol. Biol. 262:732 (1996)), each of which is incorporated herein by reference in its entirety.

Typically, CDRs form a loop structure, which can be classified as a canonical structure. The term canonical structure refers to the backbone conformation adopted by the antigen-binding loops (CDRs). As a result of comparative structural studies, five of the six antigen-binding loops were found to have only a limited set of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies may have very similar three-dimensional structures, despite the high level of amino acid sequence variability in most regions of the loops (Chothia et al., Canonical Structures For the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901 (1987) ; Chothia et al., Conformations of Immunoglobulin Hypervariable Regions, I 342:877 (1989); Martin and Thornton, Structural Families in Loops of Homologous Proteins: Automatic Classification, Modeling and Application to Antibodies, J. Mol. Biol. 263:800 (1996)), each of which is incorporated by reference in its entirety. Moreover, there is a certain relationship between the formed loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the chain length and the amino acid residues located at key positions in the loop, as well as within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of such key amino acid residues.

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The terms specifically bind, or specifically bind, or derivatives thereof, when used with antibodies and antibody fragments, represent binding, through domains encoded by immunoglobulin genes or gene fragments, to one or more epitopes of the protein of interest and, preferably, the absence of binding to other molecules in the sample. containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1 x ^10'8 M, as measured by a surface plasmon resonance or cell binding assay. In a preferred embodiment, binding specificity is measured using biolayer interferometry. Phrases such as [antigen]specific antibody mean that said antibody specifically binds to said antigen.

The term polynucleotide, which is synonymous with the term nucleic acid molecule, nucleotides or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. Additionally, the term polynucleotide refers to three-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with the backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and atypical bases such as inosine. Various modifications can be made to DNA and RNA; therefore, the term polynucleotide includes the chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA found in viruses and cells. The term polynucleotide also includes relatively short chains of nucleic acids, often called oligonucleotides.

A vector is a replicon, such as a plasmid, phage, cosmid or virus, into which another nucleotide segment can be operably inserted so that replication or expression of the segment occurs.

As used herein, the term host cell can refer to any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term host cell refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such cell. The descendants of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental effects that may occur in subsequent generations, or integration of the nucleic acid molecule into the genome of the host cell. The terms expression and production are used interchangeably herein to refer to the biosynthesis of a gene product. These terms cover the transcription of a gene into RNA. These terms also cover the translation of RNA into one or more polypeptides and further cover all naturally occurring post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof may occur within the cytoplasm of a cell or in an extracellular environment, such as a cell culture growth medium. The meaning of a term essentially the same may differ depending on the context in which it is used. Because variation is possible in the natural sequence of the light and heavy chains and the genes encoding them, it is expected that a certain level of variation within the amino acid sequences or genes encoding the antibodies or antigen-binding fragments described herein may be encountered with little or no effect on them. unique binding properties (e.g. specificity and affinity). This expectation is partly due to the degeneracy of the genetic code, as well as the evolutionary success of conservative variants of amino acid sequences that do not significantly change the nature of the encoded protein. Accordingly, in the context of nucleotide sequences, essentially the same means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identical, more preferably at least 97% identical, more preferably at least 98% identical, and more preferably at least 99% or more identical. Percent identity between two

- 7 044007 sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap introduced for optimal alignment two sequences. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm described in E. Meyers and W. Miller, Comput. Application Biosci 4, 11-17 (1988), which is built into the ALIGN program (version 2.0), using the PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage identity of two amino acid sequences can be determined using the algorithm , described in Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation possible within the amino acid sequence of a protein without significantly affecting the function of the protein is much lower than in a nucleotide sequence, since the principles of degeneracy do not apply to amino acid sequences. Accordingly, in the context of an antibody or antigen binding fragment, substantially the same refers to an antibody or antigen binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity described antibodies or antigen-binding fragments. Other embodiments include anti-FN3 domain antibodies or antigen binding fragments that have a framework, backbone, or other non-binding regions that do not share significant identity with the anti-FN3 domain antibodies and antigen binding fragments described herein, but necessarily include one or more CDRs or other sequences necessary to effect binding that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences described herein.

As used herein, the term chimeric antigen receptor (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds an antigen or target, a transmembrane domain, and an intracellular signaling domain that activates an intracellular T cell receptor. Interaction of the extracellular domain of the CAR with the target antigen on the surface of the target cell results in CAR clustering and delivers the activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis, and/or production of molecules that can mediate cell death of the target antigen-expressing cell in a major histocompatibility complex (MHC)-independent manner.

As used herein, the term extracellular antigen-binding domain, extracellular domain, or extracellular ligand-binding domain refers to the portion of a CAR that is located outside the cell membrane and is capable of binding to an antigen, target, or ligand.

As used herein, the term hinge region refers to the portion of a CAR that connects two adjacent domains of a CAR protein, for example, an extracellular domain and a transmembrane domain.

As used herein, the term transmembrane domain refers to the portion of the CAR that extends across the cell membrane and anchors the CAR to the cell membrane.

As used herein, the term intracellular signaling domain, T cell receptor activating domain, cytoplasmic signaling domain, or intracellular signaling domain refers to the portion of the CAR that is located within the cell membrane and is capable of effector signal transduction.

As used herein, the term stimulatory molecule refers to a molecule expressed by a T cell that provides the primary cytoplasmic signal sequence(s) that regulates the primary activation of the T cell receptor (TCR) complex in the stimulatory pathway for at least some aspect of the T cell signaling pathway. Stimulatory molecules include two distinct classes of cytoplasmic signal sequence: those that initiate antigen-dependent primary activation (termed primary signaling domains) and those that act in an antigen-independent manner to provide a secondary costimulatory signal (termed costimulatory signaling domains).

As used herein, the term expression refers to the biosynthesis of a gene product. This term covers the transcription of a gene into RNA. The term also covers the translation of RNA into one or more polypeptides and, in addition, covers all naturally occurring post-transcriptional and post-translational modifications. The expressed FN3 or CAR domain may be located in the cytoplasm of the host cell, in an extracellular environment such as cell culture media, or attached to the cell membrane.

As used herein, the term immune cell or immune effector cell refers to a cell that participates in an immune response, for example, in stimulating an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. In specific embodiments, the engineered immune cells are T cells and are referred to as CAR T cells because they are engineered to express the CAR of the invention.

As used herein, the term engineered immune cell refers to an immune cell, also called an immune effector cell, that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the cell's general genetic material. In embodiments of the present invention, engineered immune cells have been genetically modified to express a CAR targeting the FN3 domain in accordance with the invention.

As used herein, the term carrier refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation or other material well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term pharmaceutically acceptable carrier refers to a non-toxic material that does not adversely affect the effectiveness of the composition of the present invention or the biological activity of the composition of the present invention.

As used herein, the term subject refers to an animal, preferably a mammal. In certain embodiments, the subject is a mammal, including a non-primate mammal (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, or mouse), or primates (eg monkey, chimpanzee or human). In specific embodiments, the subject is a human.

As used herein, the term therapeutically effective amount refers to the amount of an active ingredient or component that produces a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and routinely depending on the intended purpose.

As used herein, the terms treat, treat, and cure refer to the alleviation or return to baseline of at least one measurable physical parameter associated with cancer or autoimmunity that is not necessarily apparent in the patient, but may be apparent in the patient. The terms treat, treater, and treatment may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, the terms treat, treat and treatment refer to alleviating, preventing the development or occurrence or reducing the duration of one or more symptoms associated with a disease, disorder or condition, such as a tumor or more preferably cancer. In a specific embodiment, the terms treat, treater, and treatment refer to the prevention of recurrence of a disease, disorder, or condition. In a specific embodiment, the terms treat, treater, and treatment refer to improving the survival of a patient having a disease, disorder, or condition. In a specific embodiment, the terms treat, treater, and treatment refer to treating a disease, disorder, or condition in a patient.

FN3 Domain Libraries

Tencon is a non-naturally occurring FN3 domain designed from a consensus sequence of fifteen FN3 domains of human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; US2010/0216708). The Tencon crystal structure shows six surface-exposed loops connecting the seven beta strands characteristic of FN3 domains, with the beta strands designated A, B, C, D, E, F, and G, and the loops designated AB, BC, CD , DE, EF and FG (Bork and Doolittle, PNAS USA 89:8990-8992, 1992; US6673901). These loops, or selected residues within each loop, can be randomized to create a library of FN3 domains that can be used to select new molecules that bind a particular antigen. Therefore, as described herein, a non-randomized region of the FN3 domain refers to a region within the FN3 domain that is conserved across all FN3 domains. Table 2 shows the positions and sequences of each loop and beta strand in Tencon (SEQ ID NO: 33).

- 9 044007

table 2

Domain FN3 Tencon (SEQ ID NO: 33) Heavy A 1-12 Loop AB 13-16 Straight B 17-21 Sun loop 22-28 Straight C 29-37 CD loop 38-43 Heavy 44-50 Loop DE 51-54 Straight E 55-59 Hinge EF 60-64 Straight F 65-74 Hinge FG 75-81 Tie G 82-89

Thus, libraries generated from the Tencon sequence may have randomized sequence in one or more loops or strands. For example, Tencon-based libraries may have randomized sequence in one or more of the AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop. For example, the Tencon BC loop is 7 amino acids long, so a Tencon sequence-based library with BC loop diversification can be randomized to 1, 2, 3, 4, 5, 6, or 7 amino acids. The Tencon CD loop is 6 amino acids long, so a Tencon sequence-based library with CD loop diversification can be randomized to 1, 2, 3, 4, 5, or 6 amino acids. The Tencon EF loop is 5 amino acids long, so a Tencon sequence-based library with EF loop diversification can be randomized to 1, 2, 3, 4, or 5 amino acids. The Tencon FG loop is 7 amino acids long, so a Tencon sequence-based library with FG loop diversification can be randomized to 1, 2, 3, 4, 5, 6, or 7 amino acids. Additional loop diversity in Tencon libraries can be achieved by inserting and/or deleting residues in the loops. For example, loops BC, CD, EF and/or FG can be extended by 1-22 amino acids or shortened by 1-3 amino acids. The FG Tencon loop is 7 amino acids long, whereas the corresponding loop in antibody heavy chains ranges from 4 to 28 residues. To provide maximum diversity, the FG loop can be diversified in sequence as well as in length to match the antibody CDR3 length range of 4-28 residues. For example, the FG loop can be further diversified in length by extending the loop by an additional 1, 2, 3, 4, or 5 amino acids.

Libraries designed from the Tencon sequence may also have alternative randomized surfaces formed lateral to the FN3 domain and containing two or more beta strands and at least one loop. One such alternative surface is formed by amino acids in the beta strands C and F and in the loops CD and FG (C-CD-F-FG surface). The configuration of the library based on the alternative C-CD-F-FG Tencon surface is described in US2013/0226834. Libraries designed from the Tencon sequence also include libraries designed from Tencon variants, such as Tencon variants having substitutions at positions of residues 11, 17, 46 and/or 86 (residue numbering corresponds to SEQ ID NO: 33), and such variants demonstrate increased thermal stability. Exemplary variants of Tencon are described in US 2011/0274623 and include Tencon27 (SEQ ID NO: 34) having substitutions E11R, L17A, N46V and E86I compared to Tencon of SEQ ID NO: 33.

Tencon-based libraries and other FN3 sequence-based libraries can be randomized at selected residue positions using a random or specific set of amino acids. For example, variants in a library that has random substitutions can be generated using NNK codons that encode all 20 naturally occurring amino acids. Other diversification schemes can use DVK codons to encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly and Cys. Alternatively, NNS codons can be used to obtain all 20 amino acid residues while reducing the frequency of stop codons. Libraries of FN3 domains with a biased distribution of amino acids at diversifying positions can be synthesized, for example, using Slonomics® technology (http:_//www_sloning_com). This technology uses a library of pre-formed double-stranded triplets that serve as universal building blocks sufficient for the synthesis processes of thousands of genes. The triplet library contains all possible combinations of sequences necessary to construct any desired DNA molecule. The well-known IUB code is used to designate codons.

Antibodies to the FN3 domain and antigen-binding fragments

Disclosed herein are isolated monoclonal antibodies or antigen binding fragments that specifically bind to a non-randomized region of FN3 domains. The general structure of an antibody molecule contains an antigen-binding domain, which includes heavy and light chains, and an Fc domain, which performs various functions, including complement fixation and binding to antibody receptors.

Described FN3 domain-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin structure. The disclosed antibodies or antigen binding fragments also include the IgY isotype essentially found in chicken or turkey serum and in the yolk of chicken or turkey eggs.

FN3 domain-specific antibodies and antigen-binding fragments can be obtained from any species using recombinant methods. For example, the antibodies or antigen binding fragments may be murine, rat, goat, equine, porcine, bovine, chicken, rabbit, camel, donkey, human or chimeric variants thereof. For the purpose of administration to humans, antibodies or antigen-binding fragments obtained from non-human sources can be genetically or structurally modified to be less antigenic when administered to a human patient.

In some embodiments, the antibodies or antigen binding fragments are chimeric. As used herein, the term chimeric means an antibody or antigen-binding fragment thereof comprising at least some portion of at least one variable domain derived from the amino acid sequence of a non-human mammal, rodent or reptile antibody, while the remaining portions of the antibody or antigen-binding fragment thereof fragments are of human origin.

In some embodiments, the antibodies are humanized antibodies. Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) containing a minimal sequence derived from a non-human animal immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the complementarity determining region of a non-human species (donor antibody), such as a mouse, rat or rabbit having the desired specificity, affinity and ability. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to those in an immunoglobulin from a non-human animal, and all or substantially all of the frame regions correspond to those of the human immunoglobulin sequence. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin.

The antibodies or antigen binding fragments described herein can take various forms but will include one or more antibody CDRs as shown in Table. 1.

Described herein are isolated antibodies and antigen binding fragments that specifically bind to FN3 domains. In some embodiments, FN3 domain-specific antibodies or antigen-binding fragments are obtained from rabbits. Although the FN3 domain-specific antibodies or antigen-binding fragments exemplified herein are derived from rabbit, the exemplified antibodies or antigen-binding fragments can be chimerized.

In some embodiments, an FN3 domain-specific antibody or antigen-binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. 1, and a light chain containing CDR1, CDR2 and CDR3 of any of the antibodies described in table. 1.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 4, a heavy chain CDR3 comprising SEQ ID NO: 7, a light chain CDR1, comprising SEQ ID NO: 9, a light chain CDR2 comprising SEQ ID NO: 11, and a light chain CDR3 comprising SEQ ID NO: 13. This FN3 domain specific antibody or antigen binding fragment may contain non-rabbit framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same

- 11 044007 or identical to SEQ ID NO: 14, and a light chain variable region substantially the same or identical to SEQ ID NO: 15. The described anti-FN3 domain antibodies or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 8, a light chain CDR1, comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 12, and a light chain CDR3 comprising SEQ ID NO: 13. This FN3 domain-specific antibody or antigen binding fragment may contain non-rabbit framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 14 and a light chain variable region substantially the same or identical to SEQ ID NO: 15. Antibodies Disclosed to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 6, a heavy chain CDR3 comprising SEQ ID NO: 8, a light chain CDR1, comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 12, and a light chain CDR3 comprising SEQ ID NO: 13. This FN3 domain-specific antibody or antigen binding fragment may contain non-rabbit framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 14 and a light chain variable region substantially the same or identical to SEQ ID NO: 15. Antibodies Disclosed to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 44, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 46, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 74 and a light chain variable region substantially the same or identical to SEQ ID NO: 75. Antibodies described to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 74 and a light chain variable region substantially the same or identical to SEQ ID NO: 74

- 12 044007 the same or identical to SEQ ID NO: 75. The described anti-FN3 domain antibodies or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 37, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 74 and a light chain variable region substantially the same or identical to SEQ ID NO: 75. Antibodies described to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 51, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 54, light chain CDR1 having the amino acid sequence of SEQ ID NO: 56, light chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 60. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 78 and a light chain variable region substantially the same or identical to SEQ ID NO: 79. Antibodies described to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 52, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 55, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 57, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 59, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CAR domains containing FN3. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 78 and a light chain variable region substantially the same or identical to SEQ ID NO: 79. Antibodies described to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

In some embodiments, the FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 53, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 55, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 57, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 59, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61. This is an FN3 domain-specific antibody or its the antigen binding fragment may contain non-murine framework sequences. This FN3 domain-specific antibody or antigen-binding fragment thereof can bind to non-randomized regions of the FN3 domain, can detect the expression of chimeric antigen receptors (CARs) containing FN3 domains on the surface of T cells, and can activate a T cell expressing CARs containing FN3 domains. In some embodiments, FN3 domain-specific antibodies and antigen binding fragments comprise a heavy chain variable region substantially the same or identical to SEQ ID NO: 78 and a light chain variable region substantially the same or identical to SEQ ID NO: 79. Antibodies described to the FN3 domain or antigen binding fragments can be used to produce CARs containing the described antigen binding fragments.

Also described are isolated polynucleotides encoding antibodies or antigen-binding fragments that specifically bind to the FN3 domain. Isolated polynucleotides capable of encoding the variable domain segments provided herein can be incorporated into the same or different vectors for the production of antibodies or antigen-binding fragments.

Polynucleotides encoding recombinant antigen-binding proteins are also included within the scope of the description. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, without limitation, a restriction site or a translation initiation site.

The FN3 domain-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described FN3 domain-specific antibodies or antigen-binding fragments. . Such variants may include: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or removed from the polypeptide, (c) variants in wherein one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, a protein tag, or other chemical functional group capable of conferring beneficial properties on the polypeptide, such as an epitope for an antibody, polyhistidine sequence, biotin residue, etc. Antibodies or antigen binding fragments described herein may include variants in which amino acid residues of one type are replaced by corresponding residues of another type (at conserved or non-conserved positions). In other embodiments, amino acid residues at non-conserved positions are replaced with conservative or non-conservative residues. Techniques for obtaining such variants, including genetic (deletions, mutations, etc.), chemical and enzymatic, are known to those skilled in the art.

The FN3 domain-specific antibodies or antigen-binding fragments described herein can be of several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgG. The specificity of an antibody or antigen-binding fragment thereof is determined in large part by the amino acid sequence and the location of the CDRs. Therefore, CDRs from one isotype can be transferred to another isotype without changing antigen specificity. In an alternative embodiment, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without changing antigen specificity. Accordingly, such antibody isotypes are included within the scope of the disclosed antibodies or antigen binding fragments.

The affinity of the disclosed FN3 domain-specific antibodies or antigen-binding fragments can be determined by a variety of methods known in the art, such as surface plasmon resonance or ELISA-based methods. SPR affinity assays include assays performed using the BIAcore 3000 instrument, where the assay is performed at room temperature (e.g., at or near 25°C) and antibodies capable of binding to FN3 domains are captured on the sensor. BIAcore chip using an anti-Fc antibody (e.g., goat human IgG Fc-specific antibody from Jackson ImmunoResearch laboratories, product no. 109-005-098) at approximately 75 pfu, followed by association and dissociation data collection at a flow rate of 40 µl /min.

Methods for detecting FN3 domains

Provided herein are methods for detecting FN3 domains in a biological sample by contacting the sample with an antibody or antigen binding fragment thereof described herein. As described herein, the sample can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissues (eg, surgical excised tumor tissue, biopsy materials, including those obtained using fine-needle aspiration biopsy), histological preparations, etc. In some embodiments, the described methods involve detecting FN3 domains in a biological sample by contacting the sample with any of the FN3 domain-specific antibodies or antigen binding fragments thereof described herein.

In some embodiments, a sample can be contacted with more than one of the FN3 domain-specific antibodies or antigen-binding fragments described herein. For example, a sample may be contacted with a first FN3 domain-specific antibody or antigen-binding fragment thereof and then contacted with a second FN3 domain-specific antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are not the same antibody or antigen-binding fragment. In some embodiments, the first antibody or antigen-binding fragment thereof may be attached to a surface, such as the surface of a multiwell plate, chip, or similar substrate, before being contacted with a sample. In other embodiments, the first antibody or antigen-binding fragment thereof may not be attached or fixed to anything before contacting the sample.

The disclosed FN3 domain-specific antibodies and antigen-binding fragments can be detectably labeled. In some embodiments, labeled antibodies and antigen binding fragments may facilitate detection of FN3 domains through the methods described herein. Many such marks are well known to those skilled in the art. For example, suitable tags include, but are not limited to, radioactive tags, fluorescent tags, epitope tags, biotin, chromophore tags, ECL tags, or enzymes. More specifically, the tags described include ruthenium, ¹ⁿ In-DOTA, In-diethylenetriamine pentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexa Fluor® dyes, etc.

The described FN3 domain-specific antibodies and antigen-binding fragments can be used in a variety of assays to detect FN3 domains in a biological sample. Some acceptable assays include, but are not limited to, Western blot, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry, or ELISA.

FN3 Domain Discovery Kits

This document provides kits for detecting FN3 domains in a biological sample. These kits include one or more FN3 domain-specific antibodies described herein, or antigen binding fragments thereof, and instructions for use of the kit.

The proposed FN3 domain-specific antibody or antigen-binding fragment may be in solution; be lyophilized; attached to a substrate, carrier or tablet; or may be detectably labeled.

The described kits may also include additional components used to carry out the methods described herein. By way of example, kits may contain means for obtaining a sample from a subject, a control or a reference sample, such as a sample from a subject with slowly progressing cancer and/or a subject without cancer, one or more sample compartments, and/or instructional materials describing the implementation of the methods inventions, and tissue-specific controls or standards.

The means for determining the level of FN3 domains may further include, for example, buffers and other reagents for use in the assay for determining the level of FN3 domains. The instructions may be, for example, printed instructions for performing the assay and/or instructions for assessing the expression level of FN3 domains.

The described kits may also include means for isolating a sample from a subject. These means may contain one or more pieces of equipment or reagents that can be used to obtain fluid or tissue from a subject. The means for obtaining a sample from a subject may also be means for isolating blood components, such as serum, from the blood sample. Preferably, the kit is intended for use in a human subject.

FN3 domain-targeted chimeric antigen receptors (CARs)

In other general aspects, the invention provides an FN3 domain-targeted CAR comprising an FN3 domain-specific scFv.

In one aspect, the invention relates to a CAR containing

a) an extracellular domain having an scFv that specifically binds to a non-randomized region of the FN3 domain;

b) transmembrane domain; And

c) intracellular signaling domain.

In some embodiments, in the resulting CAR, an extracellular domain is preceded by

- 15 044007 signal peptide at the N-terminus. Any suitable signal peptide can be used in the present invention. The signal peptide can be obtained from a natural, synthetic, semi-synthetic or recombinant source.

In accordance with some embodiments of the invention, the extracellular domain of the CAR contains a scFv that specifically binds to a non-randomized region of the FN3 domain. In the extracellular domain of the CAR, any scFv that specifically binds to the FN3 domain can be used in accordance with embodiments of the invention, including, but not limited to, those described herein.

In accordance with some embodiments of the invention, the CAR may further comprise a hinge region connecting the extracellular domain and the transmembrane domain. The hinge region functions to move the extracellular domain away from the surface of the engineered immune cell to ensure proper cell/cell contact, target or antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). Any suitable hinge region may be used in the CAR of the invention. It can be obtained from a natural, synthetic, semi-synthetic or recombinant source. In some embodiments, the CAR hinge region is a 6x GS peptide (SEQ ID NO: 84), or a fragment thereof, or a hinge region from a CD8 protein, or a derivative thereof. In specific embodiments, the hinge region has an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, preferably the amino acid sequence of SEQ ID NO: 24.

Any suitable transmembrane domain may be used in the CAR of the invention. The transmembrane peptide can be obtained from a natural, synthetic, semi-synthetic or recombinant source. In some embodiments, the transmembrane domain is a transmembrane domain from molecules such as CD8, CD28, CD4, CD2, GMCSFR, and the like. In specific embodiments, the transmembrane domain has an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, preferably the amino acid sequence of SEQ ID NO: 25.

Any intracellular signaling domain can be used in the CAR of the invention. In particular embodiments, the entire intracellular signaling domain is used. In other specific embodiments, a truncated portion of a signaling domain is used that transduces an effector signal. In accordance with some embodiments of the invention, the intracellular signaling domain generates a signal that stimulates the immune effector function of a cell containing a CAR, such as a CAR-T cell, including, but not limited to, proliferation, activation and/or differentiation. In specific embodiments, the signal stimulates, for example, cytolytic activity, helper activity, and/or cytokine secretion by the CAR-T cell. In other embodiments, the CAR of the invention does not use an intracellular signaling domain, and a CAR containing a scFv that specifically binds to the FN3 domain of the invention is used in conjunction with the FN3 domain to target the effector cell to target cells.

In accordance with some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from CD3-zeta, TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD16, CD22, CD27, CD28, CD30, CD79a, CD79b, CD134 (also known as TNFRSF4 or OX-40), 4-1BB (CD137), CD278 (also known as ICOS), FcsRI, DAP10, DAP12, ITAM domains or CD66d and the like.

In accordance with some embodiments, the intracellular signaling domain comprises a primary signaling domain and one or more co-stimulatory signaling domains.

In one embodiment, the intracellular signaling domain comprises a primary intracellular signaling domain having a functional signaling domain derived from human CD3 zeta. In specific embodiments, the primary intracellular signaling domain has an amino acid sequence that is at least 90% identical to SEQ ID NO: 27, preferably the amino acid sequence of SEQ ID NO: 27.

In accordance with some embodiments, the intracellular signaling domain further comprises a co-stimulatory intracellular signaling domain derived from human 4-1BB. In specific embodiments, the co-stimulatory intracellular signaling domain has an amino acid sequence at least 90% identical to SEQ ID NO: 26, preferably the amino acid sequence of SEQ ID NO: 26.

In one embodiment, the CAR of the invention is associated with a host cell expressing the CAR.

In another embodiment, the CAR of the invention is present in the isolated cell membrane of a host cell expressing the CAR.

In yet another embodiment, the CAR of the invention is purified or isolated from other components of a host cell expressing the CAR.

- 16 044007

Polynucleotides, vectors and host cells

In other general aspects, the invention relates to isolated polynucleotides and vectors encoding antibodies to the FN3 domain or CAR of the invention, and to recombinant cells containing the vectors.

Nucleic acids encoding any of the various proteins or polypeptides disclosed herein can be synthesized chemically or using other methods in the art related to this description. Codon preference can be selected to improve expression in the cell. This codon preference will depend on the cell type chosen. Specialized codon preference patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells. See, for example: Mayfield et al., PNAS USA. 2003 100(2):438-42; Sinclair et al. Protein Expr Purif. 2002(1):96105; Cornell, Curr Opin Biotechnol. 2001(5):446-9; Makrides et al. Microbiol Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

General techniques for manipulating nucleic acids are within the competence of one skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, and updates from time to time, which are incorporated herein by reference. The protein-encoding DNA is operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional transcriptional control operator sequence, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control transcription and translation termination. An origin of replication, typically providing the ability to replicate in the host, and a selection gene, facilitating the recognition of transformants, are additionally included. Suitable control elements are well known in the art.

Those skilled in the art will appreciate that the coding sequence of a protein can be changed without changing the amino acid sequence of the protein. Accordingly, those skilled in the art will appreciate that the nucleic acid sequences encoding the FN3 or CAR domain antibodies of the invention can be altered without altering the amino acid sequences of the proteins.

In one embodiment, the invention provides a vector containing an isolated nucleic acid encoding an antibody to the FN3 domain or CAR of the invention. Any vector known to those skilled in the art in light of this disclosure may be used, such as a plasmid, cosmid, phage vector, or viral vector. In one embodiment, the vector is an expression vector containing a polynucleotide sequence encoding an FN3 or CAR domain of the invention operably linked to a promoter sequence, optionally one or more other regulatory sequences.

In another embodiment, the invention relates to transient expression of a CAR according to the invention using mRNA encoding the CAR. In one aspect, the mRNA encoding a CAR is introduced into an immune effector cell as a form of transient transfection, wherein expression of the unintegrated transgene occurs over a period of time expressed in hours, days, or weeks, wherein the time period for expression is less than the time period for expression of the gene. integrated into the genome or contained in a stable plasmid replicon in the host cell. In one aspect, the mRNA is produced by in vitro transcription using a PCR-generated template.

In another general aspect, the invention provides a host cell containing an isolated nucleic acid encoding an anti-FN3 or CAR domain antibody of the invention. A host cell can be stably or transiently transfected with a nucleic acid molecule according to the invention.

Suitable host cells include prokaryotes, yeast, mammalian cells or bacterial cells. Suitable bacteria include gram-negative or gram-positive organisms, for example, E. coli or Bacillus spp. Yeast, preferably from Saccharomyces species such as S. cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Baculovirus systems for producing heterologous proteins in insect cells are discussed in Luckow and Summers, (Bio/Technology, 6:47, 1988). In some cases it is desirable to produce proteins in vertebrate cells, for example for glycosylation, and propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of suitable mammalian host cell lines include endothelial cells, monkey kidney cells COS-7, CV-1, L, C127, 3T3 cells, Chinese hamster ovary (CHO) cells, human embryonic kidney cells, HeLa cells, 293, 293T cell lines and BHK.

The host cell of the invention may be an engineered immune cell targeting the FN3 domain, which is described in detail below.

- 17 044007

Getting Protein

In another general aspect, the invention relates to a method for producing an anti-FN3 domain antibody according to the invention, comprising culturing a host cell containing a nucleic acid encoding an anti-FN3 domain antibody under conditions for producing an anti-FN3 domain antibody according to the invention, and isolating the anti-FN3 domain antibody from the cell or cell culture (eg from supernatant). Expressed antibodies to the FN3 domain can be collected from cells or cell culture and purified in accordance with conventional techniques known in the art, taking into account the present description.

In another general aspect, the invention relates to a method for producing a CAR according to the invention, comprising culturing a host cell containing a nucleic acid encoding a CAR under the conditions for producing a CAR according to the invention, and isolating the CAR. Expressed CARs can be harvested from cells and purified according to conventional techniques known in the art and described herein.

Host cells are transformed with expression or cloning vectors for protein production and cultured in conventional culture media suitably modified, for example, to induce promoters, select transformants, or amplify genes encoding desired sequences.

The host cells used to produce the proteins of this invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al, Meth. Enz. 58:44 (1979); Barnes et al, Anal. Biochem. 102:255 (1980); US 4767704; US 4657866; US 4927762; US 4560655; US 5122469; WO 90/03430; WO 87/00195 or US RE30985, can be used as a culture medium for host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides ( such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace minerals (defined as inorganic compounds typically present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary additives may also be included in suitable concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used for the host cell selected for expression and will be apparent to one skilled in the art.

The proteins disclosed herein can also be produced using in vitro cellular translation systems. For such purposes, the nucleic acids encoding the proteins must be modified to allow in vitro transcription to produce the mRNA and to allow cell-free translation of the mRNA in the particular cell-free system used. Exemplary cell-free eukaryotic translation systems of eukaryotic cells include, for example, cell-free translation systems based on mammalian or yeast cells, and exemplary cell-free prokaryotic translation systems include, for example, cell-free translation systems based on bacterial cells.

The proteins disclosed herein can also be prepared by chemical synthesis (eg, using the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL). Protein modifications can also be made by chemical synthesis.

The proteins disclosed herein can be purified using protein isolation/purification methods generally known in the art of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (eg, ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combination thereof. After protein purification, the buffers can be exchanged for various buffers and/or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

The purified proteins are preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure.

Engineered T cells with CARs targeting the FN3 domain, compositions and methods of use thereof

In another general aspect, the invention relates to engineered immune cells targeting the FN3 domain containing a CAR targeting the FN3 domain of the invention, methods for producing engineered immune cells, compositions containing engineered immune cells, and methods of using engineered immune cells to treat such diseases such as multiple myeloma.

- 18 044007

In one general aspect, the invention relates to engineered immune cells containing CARs targeting the FN3 domain according to the invention.

In accordance with some embodiments, the immune cell can be made less allogeneic, for example, by inactivating at least one gene expressing one or more T cell receptor (TCR) components, as described in WO 2013/176915, which can be combined with inactivation a gene that encodes or regulates the expression of the HLA-I/beta-2 microglobulin (B2M) protein. Accordingly, the risk of graft-versus-host disease and graft rejection is significantly reduced. A T cell that lacks a functional TCR, called a TCR knockout or TCR-KO cell, can be engineered so that it does not express any functional TCR on its surface, engineered so that it does not express one or more subunits that contain a functional TCR (e.g., designed such that it does not express (or exhibits reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon and/or TCR zeta) or is designed so that it produces very little functional TCR on its surface. Alternatively, the T cell may express a substantially impaired TCR (ie, a TCR that will not elicit an adverse immune response in the host), for example, by expressing mutated or truncated forms of one or more TCR subunits. Modified T cells that do not express a functional TCR and/or B2M can be generated by any suitable method, including knocking out or knocking down one or more TCR and/or B2M subunits. For example, using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), megaTAL, meganuclease or zinc finger endonuclease (ZFN) can achieve knockdown in a T cell TCR and/or B2M.

In specific embodiments, the immune effector cell containing the CAR targeting the FN3 domain of the invention is a T cell, NKT cell or NK cell, preferably a human T cell or human NK cell, more preferably a cell with a TCR knockout cell, most preferably a human TCR knockout cell and/or an HLAI/B2M knockout cell. In other embodiments, the immune effector cell containing the CAR targeting the FN3 domain of the invention is an engineered T cell line, such as the TALL-104 T cell line (i.e., IL-2-dependent human unrestricted cytotoxic T cell line that expresses CD8 and CD3, but not CD16).

Immune effector cells according to the invention can be autologous (that is, their own, for example, autogenous) or non-autologous and (that is, not their own, for example, allogeneic, syngeneic, xenogeneic). Autologous refers to any material obtained from the same individual to whom it will subsequently be administered. Non-autologous refers to any material obtained from another individual of the same species as the individual to whom the material will subsequently be administered.

In another general aspect, the invention relates to methods of producing engineered immune cells targeting the FN3 domain containing CARs targeting the FN3 domain according to the invention. The vector encoding the CAR can be directly transduced into an immune cell. Alternatively, in vitro transcribed RNA or synthetic RNA encoding the CAR can be introduced into an immune cell.

In accordance with specific embodiments, a method of producing engineered immune cells targeting the FN3 domain comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs in accordance with embodiments of the invention. Methods for preparing immune cells for immunotherapy are described, for example, in WO 2014/130635, WO 2013/176916 and WO 2013/176915, which are incorporated herein by reference. Specific steps that can be used to produce engineered immune cells are disclosed, for example, in WO 2014/039523, WO 2014/184741, WO 2014/191128, WO 2014/184744 and WO 2014/184143, which are incorporated herein by reference.

In a specific embodiment, immune effector cells, such as T cells, are genetically modified by a CAR according to the invention (eg, transduced with a viral vector containing a nucleic acid encoding the CAR) and then activated and propagated in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR using methods as described, for example, in US 6,352,694, US 6,534,055, US 6,905,680, US 6,692,964, US 5,858,358, US 6,887,466, US 6,905,681. US 7144575, US 7067318, US 7172869, US 7232566, US 7175843, US 5883223, US 6905874, US 6797514, US 6867041, US 2006/121005, which are incorporated herein by reference. T cells can be expanded in vitro or in vivo. In general, T cells of the invention can be propagated by contacting a surface containing a substance attached thereto that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface

- 19 044007

T cells. As non-limiting examples, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with an activator protein kinase C (eg, bryostatin) in combination with a calcium ionophore, by activating the CAR itself. To costimulate an accessory molecule on the surface of T cells, a ligand is used that binds the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. Conditions suitable for culture of T cells include, for example, suitable media (eg, minimal essential medium or RPMI 1640 or Xvivo 5 (Lonza)), which may contain factors necessary for proliferation and viability, including serum (eg, fetal bovine or human serum), cytokines such as IL-2, IL-7, IL-15 and/or IL-21, insulin, IFN-gamma, GM-CSF, TGF-beta and/or any other cell growth supplements , known to a person skilled in the art. In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US 6,040,177, US 5,827,642 and WO 2012129514, which are incorporated herein by reference.

In some embodiments, a cell expressing a CAR of the invention may further comprise a second CAR having an extracellular domain that specifically binds to the same target or a different target. Preferably, the immune cell expresses two CARs that specifically bind to two different targets, or the immune cell expresses a bispecific receptor, such as a CAR, containing two FN3 domains that specifically bind to two different targets, i.e., an FN3 domain and another target associated with disease of interest. For example, another target may also be related to the type of cancer. More preferably, the two CARs also have different intracellular signaling domains, eg, the first CAR has a co-stimulatory signaling domain but not the primary signaling domain, and the second CAR has a primary signaling domain but not the co-stimulatory signaling domain, or vice versa. By placing a co-stimulatory signaling domain, such as that from 4-1BB, CD28, CD27 ICOS, or OX-40 on one CAR, and a primary signaling domain, such as that from CD3-zeta, on another CAR, one can limit CAR activity by towards cells in which both targets are expressed, for example, to increase specificity.

In some embodiments, a CAR-expressing cell of the invention may further comprise an inhibitory CAR as a self-regulating safety switch to limit T cell-based therapy and prevent off-tumor toxicity. For example, an inhibitory CAR may contain an extracellular domain that specifically binds to a target found on normal cells but not on target cancer cells. An inhibitory CAR also contains an intracellular domain having an inhibitory receptor signaling domain, such as an intracellular domain of an inhibitory molecule, including, but not limited to, PD1, PD-L1, PD-L2, CTLA4, TIM3, SEACAM (e.g., SEACAM-1, SEACAM -3 and/or SEASAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR , A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFbeta receptor. Cells expressing FN3 domain-targeting CAR and inhibitory CAR are suppressed when encountering a normal cell, but are activated when encountering a tumor cell that does not express the normal cell target.

In some other embodiments, the CAR-expressing cell of the invention may further comprise an agent that enhances the activity of the CAR-expressing cell. For example, in a host cell, the agent may inhibit the activity of an inhibitory molecule, such as those described herein.

In accordance with specific embodiments, the engineered immune cells expressing the CAR are further genetically engineered to be chemoresistant. Such chemoresistance may allow engineered immune cells to survive in the presence of drugs while selectively targeting the FN3 domain of interest.

In accordance with some embodiments, drug resistance can be provided to CAR-expressing cells by genetic engineering to express at least one drug resistance gene. The drug resistance gene of the invention may encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, antiimmunophilins, analogs or derivatives thereof, etc. Several drug resistance genes have been identified that can be used to confer drug resistance to engineered CAR-expressing immune cells according to the invention. See, for example, Takebe et al., Mol Ther. 2001 Jan;3(1):88-96.; Sugimoto et al, J Gene Med. 2003 May;5(5):366-76; Zielske et al., J Clin Invest. 2003 Nov;112(10):1561-70; Nivens et al, Cancer Chemother Pharmacol. 2004 Feb;53(2): 107-15;

- 20 044007

Bardenheuer et al., Leukemia. 2005 Dec;19(12):2281-8; Kushman et al., Carcinogenesis. 2007 Jan;28(1):207-14. Examples of drug resistance genes that may be expressed in cells include a mutant or modified form of dihydrofolate reductase (DHFR), a mutant or modified form of ionisin 5'-monophosphate dehydrogenase II (IMPDH2), multidrug resistance protein-1 (MDR1) calcineurin, methylguanine transferase ( MGMT), microRNA-21, antibiotic resistance genes ble and mcrA, etc. In certain embodiments, said drug resistance genes may be expressed in a cell by any suitable means, including, for example, introducing a transgene encoded by at least one vector into the cell.

Resistance to cancer chemotherapy can also be achieved, for example, by inactivating genes that make cells sensitive to drugs. Examples of genes that can be inactivated to confer drug resistance on cells include, for example, CD52, glucocorticoid receptors, CD3, human hypoxanthine-guanine phosphoribosyltransferase (HPRT), human deoxycytidine kinase (dCK), etc. Genes responsible for cell sensitivity to anticancer drugs can be inactivated by any suitable method, including gene knockout or knockdown, for example, using siRNA, shRNA, CRISPR, TALEN or ZFN.

In yet another general aspect, the invention relates to a pharmaceutical composition comprising an engineered immune cell targeting the FN3 domain of the invention and a pharmaceutically acceptable carrier. In light of this disclosure, any pharmaceutically acceptable carrier suitable for use in a CAR-T pharmaceutical composition may be used in this invention.

In yet another general aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.

In accordance with embodiments of the invention, a therapeutically effective amount of the pharmaceutical composition stimulates an immune response in a subject in need thereof, preferably resulting in treatment of a disease, disorder or condition; preventing or slowing the progression of a disease, disorder or condition; or reduction or complete relief of symptoms associated with an immune disease, disorder or condition.

In certain embodiments, the disease, disorder, or condition being treated is a hyperproliferative disease. In other specific embodiments, the disease, disorder, or condition being treated is a cancer or tumor or malignant hyperproliferative disease, preferably a cancer selected from the group consisting of solid tumor, hematologic malignancy, bladder cancer, biliary tract cancer , brain cancer, breast cancer, colon cancer, esophageal cancer, stomach cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer , salivary gland cancer, gastric cancer, epithelial thymus cancer and thyroid cancer.

In certain embodiments, a therapeutically effective amount of the FN3 domain-targeted immune cell composition is sufficient to achieve one, two, three, four, or more of the following effects in a subject in need thereof: (i) reducing or alleviating the severity of the disease, disorder or the condition being treated or a symptom associated therewith; (ii) shortening the duration of the disease, disorder or condition being treated, or a symptom associated therewith; (iii) preventing the progression of a disease, disorder or condition being treated, or a related symptom; (iv) causing regression of the disease, disorder or condition being treated, or a related symptom; (v) preventing the development or occurrence of a disease, disorder or condition being treated, or a related symptom; (vi) preventing the recurrence of a disease, disorder or condition being treated, or a related symptom; (vii) reducing the likelihood of hospitalization of a subject having a disease, disorder or condition being treated, or a related symptom; (viii) reducing the length of hospitalization of a subject having a disease, disorder or condition being treated, or a related symptom; (ix) increasing the survival rate of a subject with the disease, disorder or condition being treated, or a related symptom; (xi) inhibiting or suppressing the disease, disorder or condition being treated, or a related symptom in the subject; and/or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy. In specific embodiments, a therapeutically effective amount refers to an amount of drug that is sufficient to provide one, two, three, four or more of the following effects: (i) reducing tumor volume; (ii) reducing the number of tumor cells; (iii) reducing the number of metastases; (iv) increasing life expectancy; (v) reduction

- 21 044007 proliferation of tumor cells; (vi) reducing the survival of tumor cells; (vii) alleviating physiological symptoms associated with cancer; and/or (viii) preventing the occurrence of a tumor.

The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition being treated, the means of administration, the target site, the physiological condition of the subject (including, for example, age, body weight, health), whether the subject human or animal, other drugs administered and whether the treatment is prophylactic or therapeutic. Treatment dosages were optimally adjusted to optimize safety and efficacy. The exact dose can be determined by one skilled in the art using known techniques. Typically, immune cells targeting the FN3 domain are administered at a dose of about 10 to 10 cells/kg body weight. In accordance with some embodiments, a total dose of cells may be administered to a subject through dose fractionation, such as one, two, three, or more separate partial dose administrations.

In accordance with specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein may be formulated to be suitable for intravenous, subcutaneous or intramuscular administration.

In certain embodiments, a composition used in the treatment of cancer may be used in combination with another treatment, including, without limitation, chemotherapy, lymphocyte depletion therapy, radiation therapy, another immuno-oncology drug, targeted therapy, a cancer vaccine, or other anticancer drugs. .

As used herein, the term combination in the context of administering two or more drugs to a subject refers to the use of more than one therapy. Use of the term in combination does not limit the order in which the drugs are administered to a subject. For example, the first therapeutic agent (e.g., a composition described herein) may be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours , 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks, after (for example, after 5 min, 15 min, 30 min, 45 min, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) of administration of the second therapeutic agent to the patient or simultaneously with such administration.

In yet another general aspect, the invention provides a method of directing immune cells to kill FN3 domain binding cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.

In yet another general aspect, the invention relates to a method for preparing a pharmaceutical composition containing FN3 domain-targeted immune cells according to the invention, comprising combining the FN3 domain-targeted immune cells with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

The present invention also provides the following non-limiting embodiments.

Embodiments

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a non-randomized region of the fibronectin type III domain (FN3).

2. The isolated antibody or antigen binding fragment of Embodiment 1, comprising

a) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 1, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 4, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 7, Light chain CDR1 having the amino acid sequence SEQ ID NO: 9, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 13;

b) heavy chain CDR1 having the amino acid sequence SEQ ID NO: 2, heavy chain CDR2 having the amino acid sequence SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence SEQ ID NO: 8, light chain CDR1 having the amino acid sequence SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 13;

c) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 3, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 6, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 8, Light chain CDR1 having the amino acid sequence SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 13;

d) heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41, heavy chain CDR3 having

- 22 044007 amino acid sequence SEQ ID NO: 44, light chain CDR1 having the amino acid sequence SEQ ID NO: 46, light chain CDR2 having the amino acid sequence SEQ

ID NO: 48, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50;

e) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 36, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 42, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 45, Light chain CDR1 having the amino acid sequence SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50;

f) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 37, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 43, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 45, Light chain CDR1 having the amino acid sequence SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50;

g) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 38, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 51, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 54, Light chain CDR1 having the amino acid sequence SEQ ID NO: 56, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 58, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 60;

h) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 39, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 52, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 55, Light chain CDR1 having the amino acid sequence SEQ ID NO: 57, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 59, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61; or

i) Heavy chain CDR1 having the amino acid sequence SEQ ID NO: 40, Heavy chain CDR2 having the amino acid sequence SEQ ID NO: 53, Heavy chain CDR3 having the amino acid sequence SEQ ID NO: 55, Light chain CDR1 having the amino acid sequence SEQ ID NO: 57, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 59, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61.

3. The antibody or antigen binding fragment of Embodiment 1, wherein

a) the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 14, and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 15;

b) the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 74, and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 75; or

c) the antibody heavy chain variable region contains the amino acid sequence of SEQ ID NO: 78, and the antibody light chain variable region contains the amino acid sequence of SEQ ID NO: 79.

4. The antibody or antigen binding fragment of Embodiment 1, wherein

a) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 18, and the antibody light chain contains the amino acid sequence of SEQ ID NO: 19;

b) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 20, and the antibody light chain contains the amino acid sequence of SEQ ID NO: 21;

c) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 62, and the antibody light chain contains the amino acid sequence of SEQ ID NO: 63; or

d) the antibody heavy chain contains the amino acid sequence of SEQ ID NO: 64, and the antibody light chain contains the amino acid sequence of SEQ ID NO: 65.

5. The antigen binding fragment of any one of embodiments 1-4, which is a Fab fragment, a Fab2 fragment, or a single chain antibody.

6. The antibody or antigen binding fragment of any one of embodiments 15, which is an IgG.

7. An isolated polynucleotide encoding an antibody or antigen binding fragment according to any one of embodiments 1-6.

8. A vector containing the polynucleotide of embodiment 7.

9. A host cell containing the isolated polynucleotide of embodiment 7.

10. A host cell containing the vector of embodiment 8.

11. An isolated polynucleotide encoding a chimeric antigen receptor (CAR), containing:

(a) an extracellular domain containing scFv that specifically binds to a non-randomized region of the FN3 domain;

(b) transmembrane domain; and (c) an intracellular signaling domain,

- 23 044007 wherein the CAR optionally further comprises a hinge region connecting the extracellular domain and the transmembrane domain.

12. The isolated polynucleotide of embodiment 11, wherein the CAR comprises (a) an extracellular domain containing a scFv having an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 68-73;

(b) a hinge region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 24;

(c) a transmembrane domain containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 25; and (d) an intracellular signaling domain comprising a co-stimulatory domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and a primary signaling domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 : 27.

13. The isolated polynucleotide of embodiment 12, wherein (a) the extracellular domain comprises the amino acid sequence of one of SEQ ID NOs:68-73;

(b) the hinge region contains the amino acid sequence of SEQ ID NO: 24;

(c) the transmembrane domain contains the amino acid sequence of SEQ ID NO:25; and (d) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 27.

14. A vector containing the polynucleotide of any one of embodiments 11-13.

15. A host cell comprising the polynucleotide of any one of embodiments 11-13.

16. A host cell containing the vector of embodiment 14.

17. A chimeric antigen receptor (CAR) comprising (a) an extracellular domain containing an scFv that specifically binds to a non-randomized region of the FN3 domain;

(b) transmembrane domain; and (c) an intracellular signaling domain, wherein the CAR optionally further comprises a hinge region connecting the extracellular domain and the transmembrane domain.

18. The CAR of embodiment 16, comprising (a) an extracellular domain containing a scFv having an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 68-73;

(b) a hinge region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 24;

(c) a transmembrane domain containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 25; and (d) an intracellular signaling domain comprising a co-stimulatory domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and a primary signaling domain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 : 27. 19. The CAR of embodiment 17, wherein (a) the extracellular domain comprises the amino acid sequence of one of SEQ ID NOs:68-73;

(b) the hinge region contains the amino acid sequence of SEQ ID NO: 24;

(c) the transmembrane domain contains the amino acid sequence of SEQ ID NO:25; and (d) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 27.

Examples

The following examples are provided as a supplement to the above description and in order to better understand the subject matter of the invention described herein. These examples should not be considered limiting of the described subject matter of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, those skilled in the art will recognize various modifications and changes that should be included within the scope of the invention and that can be made without departing from the scope of the invention. inventions.

Example 1: Immunization of rabbits to generate antibodies against the FN3 domain.

Generation of rabbit monoclonal antibodies was performed using the Tencon25 antigen (SEQ ID NO: 28).

LPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERS

YDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

Animals: 3 month old New Zealand White rabbits with IDs E4831 and E4832 and immunization.

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Immunization protocol.

Rabbits were immunized using a standard protocol of five injections and two test blood draws per rabbit. At each injection, an aliquot of antigen was thawed and combined with complete Freund's adjuvant (CFA) (for the first injection) or incomplete Freund's adjuvant (IFA) for subsequent injections. The route of administration was subcutaneous injection (SC). Information about immunization is given in table. 3.

Table 3

Detailed description of rabbit immunization

I) antigen Sh rabbit Type number quantity Note Tepsop25 E4831 Blood collection 0 5 ml Injection 1 0.4 Injection 2 0.2 Injection 3 0.2 Blood collection 1 5 ml Injection 4 0.2 Blood collection 2 5 ml Tepsop25 E4832 Blood collection 0 5 ml Injection 1 0.4 Injection 2 0.2 Injection 3 0.2 Blood collection 1 5 ml Injection 4 0.2 Blood collection 2 5 ml Injection 5 0.4

Determination of titer using ELISA.

Anti-Tencon25 serum titer and selectivity and specificity of Tencon28 (SEQ ID NO: 29) and P114-83 (SEQ ID NO: 30) antigens were assessed using test blood draws 1 and 2 using standard Epitomics, Inc. protocols. In conclusion, both rabbits had a good immune response to the immunogen and met the standard threshold for splenectomy (OD >0.3 at 1:64k dilution). Rabbit E4832 was selected as a candidate for splenectomy and monoclonal fusion.

Splenectomy.

Rabbit E4832 received an intravenous booster injection followed by splenectomy.

Spleens were obtained and splenocytes were isolated according to standard Epitomics procedures (Table 3).

Table 4

Detailed description of splenocyte isolation

ID antigen ID a rabbit Fabric type Weight (g) Size(cm) Viability (%) Total number cells per ml Tepsop25 E4832 Spleen 2.14 6 80 1300

Merger.

Two hundred million lymphocyte cells were fused with 100 million fusion partner cells and seeded in 20X 96-well plates, respectively (Table 4). The plates were stored in tissue culture incubators under standard conditions.

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Table 5

Merger Information

Antigen ID Merge code Merger data Number of tablets Merger efficiency (%) CEN-11 F1 08/10/2011 20 46 CEN-11 F2 08/11/2011 20 59

Cell growth was examined 2-3 weeks after fusion, and fusion efficiency was calculated using the number of wells with growth divided by the total number of wells examined. At least two plates were examined for each fusion.

The screening process is briefly described below.

Preliminary screening: plates No. 5 and No. 25.

Primary screening: remaining 38 plates.

Screening method: standard ELISA, plates coated with 50 ng Tencon25/well.

Positive control: blood sample 2 from E4832 at a dilution of 1:10 thousand.

Results: 158 clones with OD greater than 0.5 were considered presumptive positive and were further expanded into a 24-well plate.

Confirmatory screening: plates coated with 50 ng Tencon25/well or coated with 50 ng Tencon28 or P114-83.

Results.

151 clones were confirmed positive for Tencon25. Among them, 78 were specific for Tencon25 because they are negative for Tencon28.

Example 2. Selection of antibodies against the FN3 domain.

Production.

The clone supernatant was assessed for binding to all centirins and for absence of binding to the negative control protein. Based on this assessment, one clone, CEN-25105-5, was selected for production. Hybridoma cells were cultured and adapted to serum-free medium and inoculated into 1-L Integra flasks. After collection, antibodies were purified using Protein A and QCed resins. The yield was 20 mg for CEN-25-105-5.

Table 6

Amino acid sequences CDR CEN-25-105-5 (SEQ ID NO:)

Defining boundaries HCCDR1 HC-CDR2 HC-CDR3 LC-CDR1 LCCDR2 LC-CDR3 IMGT GIDLSTS V (1) IYTNVNT (4) ARAVYAGA MDL (7) ERIYSN (9) KAS (11) QYTSYGSGY VGT (13) Kabat TSVMG (2) FIYTNVNTYYAS WAKG (5) AVYAGAMD L (8) QASERIYS NLA (10) KASTL AS (12) QYTSYGSGY VGT (13) Chotiya GIDLSTS (3) YTNVN (6) AVYAGAMD L (8) QASERIYS NLA (10) KASTL AS (12) QYTSYGSGY VGT (13)

VH and VL CEN-25-105-5 are shown in the table below. 7.

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Table 7

Sequences of the variable regions of the heavy and light chains of CEN-25-105-5

Type sequences Subsequence VH SEQ ID NO: Subsequence VL SEQ ID NO: Amino acid QSLEESGGRLVTPGTP LTLTCTVSGIDLSTSV MGWVRQAPGKGLESI GFIYTNVNTYYASWA KGRFTISRTSTTVDLKI TSPTTGDTATYFCAR AVYAGAMDLWGQGT LVTVSS 14 DVVMTQTPASVSGPV GGTVTIKCQASERIYS NLAWYQQKPGQPPK LLIYKASTLASGVSSR FKGSGSGTEFTLTIRD LECADAATYSCQYTS YGSGYVGTFGGGTEV VVEG 15 DNA Ctggagggagtccgggggtcgcc tggtcacgcctgggacacccctg acactcacctgcacagtctctgga atcgacctcagtacctctgtcatgg gttgggtccgccaggctccagg aaggggctggaatccatcggattc atttatactaatgttaacacatacta cgcgagctgggcaaaaggccga ttcaccat ctccagaacctcgacc acggtggatctgaaaatcaccagt ccgacaaccggggacacggcca cctatttctgtgccagagctgtttat gctggtgctatggacttgtggggc caaggcaccctggtcaccgtctcc tea 16 gatgttgtgatgacccagactcca gcctccgtgtctggacctgtggga ggcacagtcaccatcaagtgcca ggccagtgagagaatttatagca atttagcctggtatcagcagaaac c aggc agcctccc aaactcctg atctac aaggcatcc actc tggca tctggggtctcatcgcggttcaaa ggcagt ggatctggacagagtt cactctcaccatcagggaccttga gtgtgccgatgctgccacttactc ctgtcaatatacttcttatggcagtg gttatgttggtactttcggcggagg gaccgaggtggtggtcgaaggt 17

The heavy chain and light chain variable regions for CEN-25-105-5 were cloned into rat and mouse IgG-kappa expression vectors for expression and characterization. CEN-25-105-5 is currently named AS7B91 as mouse anti-kappa IgG2a antibody, and AS7B90 as rat anti-kappa IgG1 antibody. AS7B16 and AS7B82 were also derived from mouse and rabbit V regions, respectively. In table 8 and 9 show the CDR sequences and V region sequences of AS7B16 and AS7B82. The heavy and light chain sequences for all antibodies are shown in Table 1 below. 10.

Table 8

Amino acid sequences of CDRs AS7B16 and AS7B82 AS7B16

Defining boundaries HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC- CDR2 LC- CDR3 IMGT GFSLNTS GTG (35) IWWDDDK (41) VRIKGR MDY (44) QSVLFGSKQKNY (46) WAS (48) HQYLSL FT (50) Kabat TSGTGVG (36) HIWWDDDKGYN PALKS (42) IKGRMD Y(45) KSSQSVLFGSKQ KNYLA (47) WASTR ES (49) HQYLSL FT (50) Chotiya GFSLNTS GT (37) WWDDD (43) IKGRMD Y(45) KSSQSVLFGSKQ KNYLA (47) WASTR ES (49) HQYLSL FT (50)

AS7B82

Defining boundaries HCCDR1 HC-CDR2 HC-CDR3 LC-CDR1 LCCDR2 LC-CDR3 IMGT GIDFSSV AY (38) IYAGSSSSI (51) ARGLFTSGS GYYIDM (54) QSIGSD (56) SAS (58) QCTYSSSTG YNA (60) Kabat SVAYM C (39) CIYAGSSSSSIYYAS WAKG (52) GLFTSGSGY YIDM (55) QASQSIGS NLA (57) GASNL AA (59) QRGYISSAV DFFV (61) Chotiya GIDFSSV A (40) YAGSSSS (53) GLFTSGSGY YIDM (55) QASQSIGS NLA (57) GASNL AA (59) QRGYISSAV DFFV (61)

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Table 9

Sequences of the variable regions of the heavy and light chains of AS7B16 and AS7B82

Type sequences VH sequence SEQ ID NO: VL sequence SEQ ID NO: Amino acid subsequence AS7B16 QVTLKESGPGILQPSQTLS LTCSFSGFSLNTSGTGVG WIRQPS GKGLEWLAHIW WDDDKGYNPALKSRLTIS KNTSSNLVFLKIASVDTA DTATYYCVRIKGRMDYW GQGTSVTVSS 74 NIMMTQSPSSLAVSAGEK VTMNCKSSQSVLFGSKQ KNYLAWYQQKPGQSPKL LIYWASTRESGVPDRFTG SGSGTDFILTISNVQAEDL AVYYCHQYLSLFTFGSGT KLEIK 75 DNA AS7B16 AGGTTACTCTGAAAGAG TCTGGCCCTGGGATATT GCAGCCCTCCCAGACCC TCAGTCTGACTTGTTCTT TCTCTGGGTTTTCACTGA ACACTTCTGGTACGGGT GTAGGCTGGATTCGTCA GCCTTCAGGGAAGGGTC TGGAGTGGCTGGCACAC ATTTGGTGGGATGATGA CAAGGGGTATAACCCAG CCCTGAAGAGCCGACTG ASAATSTSSAAAAAAASAS CTCCAGCAACCTGGTAT TCCTCAAGATCGCCAGT GTGGACACTGCAGATAC TGCCACATATTACTGTGT TCGAATCAAAGGCCGGA TGGACTACTGGGGTCAA GGAACCTCAGTCACCGT CTCCTSA 76 AACATTATGATGACACA GTCGCCATCCTCTCTGG CTGTGTCTGCAGGAGAA AAGGTCACTATGAACTG TAAGTCCAGTCAAAGTG TTTTATTCGGTTCAAAA CAGAAGAACTATTTGGC CTGGTACCAGCAGAAAC CAGGGCAGTCTCCTAAA TTGCTGATCTACTGGGC ATCCACTAGGGAATCTG GTGTCCCTGATCGCTTC ACAGGCAGTGGATCTGG GACAGATT TTATACTTA CCATCAGCAATGTACAA GCTGAAGACCTGGCAGT TTATTACTGTCATCAAT ACCTCTCCCTATTCACGT TCGGCTCGGGGACAAAG TTGGAAATAAAA 77 Amino acid subsequence AS7B82 QEQQKESGGGLVKPGASL TLTCTASGIDFSSVAYMC WVRQAPGKGLEWIACIY AGSSSSIYYASWAKGRFT VSRTSSTTVTLQMTSLTA ADTATYFCARGLFTSGSG YYIDMWGPGTLVTVSS 78 DVVMTQTPSSVEVAVGG TVTIKCQASQSIGSNLAW YQQKPGQRPKLLIYGASN LAAGVPSRFSGSGSGTQF TLTISDVECADAATYYCQ RGYISSAVDFFVFGGGTE VVVKG 79 DNA AS7B82 CAGGAGCAGCAGAAGG AGTCCGGGGGAGGCCTG GTCAAGCCTGGGGCATC CCTGACACTCACCTGCA CAGCTTCTGGAATCGAC TTCAGTAGTGTCGCCTAC ATGTGTTGGGTCCGCCA GGCTCCAGGGAAGGGGC TGGAGTGGATCGCATGC ATTTATGCTGGTAGTAGT AGTAGCATCTACTACGC GAGCTGGGGCGAAAGGCC GATTCACCGTCTCCAGA ACCTC GTCTACCACGGT GACTCTGCAAATGACCA GTCTGACAGCCGCGGAC ACGGCCACCTATTTCTGT GCGAGAGGTCTATTTAC TAGTGGTAGTGGATATT ATATAGACATGTGGGGC CCAGGCACCCTGGTCAC CGTCTCCTCA 80 GATGTCGTGATGACCCA GACTCCATCCTCTGTGG AGGTAGCTGTGGGAGGC ACAGTCACCATCAAGTG CCAGGCCAGTCAGAGCA TTGGTAGTAATTTAGCC TGGTATCAGCAGAAACC AGGGCAGCGTCCCAAGC TCCTGATCTATGGTGCA TCCAATCTGGCCGCTGG GGTCCCATCCGCGTTCA GTGGCAGTGGATCTGGG ACACAGTTCACTCTCAC CATCA GCGACGTGGAGT GTGCCGATGCTGCCACT TACTACTGTCAACGGGG TTATATTAGCAGTGCTG TTGATTTTTTTGTTTTCG GCGGAGGGACCGAGGT GGTGGTCAAAGGT 81

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Table 10

AS7B91 heavy and light chain sequences

Clone Heavy chain amino acid sequence SEQ ID NO: Light chain amino acid sequence SEQ ID NO: AS7B91 QSLEESGGRLVTPGTPLTLT CTVSGIDLSTSVMGWVRQA PGKGLESIGFIYTNVNTYYA SWAKGRFTISRTSTTVDLKI TSPTTGDTATYFCARAVYA GAMDLWGQGTLVTVSSAK TTAPSVYPLAPVCGDTTGSS VTLGCLVKGYFPEPVTLTW NSGSLSSGVHTFPAVLQSDL YTLSSSVTVTSSTWPSQSI TC NVAHPASSTKVDKKIEPRGP TIKPCPPCKCPAPNLLGGPSV FIFPPKIKDVLMISLSPIVTCV VVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTL RVVSALPIQHQDWMSGKEF KCKVNNKDLPAPIERTISKP KGSVRAPQVYVLPPPEEEM TKKQVTLTCMVTDFMPEDI YVEWTNNGK TELNYKNTEP VLDSDGSYFMYSKLRVEKK NWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK 18 DVVMTQTPASVSGPVGGTV TIKCQASERIYSNLAWYQQK PGQPPKLLIYKASTLASGVS SRFKGSGSGTEFTLTIRDLEC ADAATYSCQYTSYGSGYVG TFGGGTEVVVEGRADAAPT VSIFPPSSEQLTSGGASVVCF LNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYS MSSTLTLTK DEYERHNSYTC EATHKTSSTSPIVKSFNRNEC 19

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AS7B90 QSLEESGGRLVTPGTPLTLT CTVSGIDLSTSVMGWVRQA PGKGLESIGFIYTNVNTYYA SWAKGRFTISRTSTTVDLKI TSPTTGDTATYFCARAVYA GAMDLWGQGTLVTVSSAET TAPSVYPLAPGTALKSNSM VTLGCLVKGYFPEPVTVTW NSGALSSGVHTFPAVLQSGL YTLTSSVTVPSSTWPSQTV T CNVAHPASSTKVDKKIVPR NCGGDCKPCICTGSEVSSVFI FPPKPKDVLTITLTPKVTCV VVDISQDDPEVHFSWFVDD VEVHTAQTRPPEEQFNSTFR SVSELPILHQDWLNGRTFRC KVTSAAFPSPIEKTISKPEGR TQVPHVYTMSPTKEEMTQN EVSITCMVKGFYPPDIYVEW QMNGQ PQENYKNTPPTMDT DGSYFLYSKLNVKKEKWQQ GNTFTCSVLHEGLHNHHTE KSLSHSPGK 20 DVVMTQTPASVSGPVGGTV TIKCQASERIYSNLAWYQQK PGQPPKLLIYKASTLASGVS SRFKGSGSGTEFTLTIRDLEC ADAATYSCQYTSYGSGYVG TFGGGTEVVVEGRADAAPT VSIFPPSSEQLASGGASVVCF INKFYPKDISVKWKIDGSER QNDVLNSVTDQDSKDSTYS MSSTLTLTK ADYERHNLYT CEVVHKTSASPVVKSFNRN EC 21 AS7B16 QVTLKESGPGILQPSQTLSLT CSFSGFSLNTSGTGVGWIRQ PSGKGLEWLAHIWWDDDK GYNPALKSRLTISKNTSSNL VFLKIASVDTADTATYYCV RIKGRMDYWGQGTSVTVSS KTTPPSVYPLAPGSAAQTNS MVTLGCLVKGYFPEPVTVT WNSGSLSSGVHTFPAVLESD LYTLSSSVTVPSSPRPSETVT CNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFP PKPKDVLTITLTPKVTCVVV DISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSV SELPIMHQDWLNGKEFKCR VNSAAFPAPIEKTISKTKGRP KAPQVYTIPPPKEQMAKDK VSLTCMITDFFPEDITVEWQ WNGQPAEN YKNTQPIMNTN GSYFVYSKLNVQKSNWEAG NTFTCSVLHEGLHNHHTEKS LSHSPGK 62 NIMMTQSPSSLAVSAGEKVT MNCKSSQSVLFGSKQKNYL AWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFIL TISNVQAEDLAVYYCHQYL SLFTFGSGTKLEIKRADAAP TVSIFPPSSEQLTSGGASVVC FLNNFYPKDINVKWKIDGSE RQNGVLNSWTDQDSKDSTY SMSSTLT LTKDEYERHNSYT CEATHKTSSTSPIVKSFNRNE C 63 AS7B82 QEQQKESGGGLVKPGASLT LTCTASGIDFSSVAYMCWV RQAPGKGLEWIACIYAGSSS SIYYASWAKGRFTVSRTSST TVTLQMTSLTAADTATYFC ARGLFTSGSGYYIDMWGPG 64 DVVMTQTPSSVEVAVGGTV TIKCQASQSIGSNLAWYQQK PGQRPKLLIYGASNLAAGVP SRFSGSGSGTQFTLTISDVEC ADAATYYCQRGYISSAVDF FVFGGGTEVVVKGRTVAAP 65

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TLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKT KPREEQYNSTY RVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSP GK SVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRG EU

Example 3. Binding of recombinant AS7B90 and AS7B91 to the FN3 domain compared to the parent rabbit antibody CEN-25-105-5.

Supernatants for AS7B90 and AS7B91, which are rat and mouse chimeras, respectively, of CEN-25-105-5, were assessed for their ability to bind recombinant FN3 domains by ELISA. Three plates were dose-dependently coated with FN3 A3 domain specific for human cMET (SEQ ID NO: 31), 83v2-ABD specific for human EGFR with albumin binding domain (SEQ ID NO: 32), tencon 25 which has no specificity (SEQ ID NO: 28), or negative control protein, all 50 μl/well in 50 mM PBS. The plates were incubated overnight at 4°C. Supernatants were discarded from the plates and 200 μl/well of Superblock was added to block the plates for 1 h at room temperature. The plates were washed 3 times with TBS-T. Supernatants and CEN25-105-5 were prepared at a concentration of 1 μg/ml in Superblock and added to the plates. The plates were incubated for 1 hour at room temperature. The plates were washed three times with TBS. Secondary antibodies (HRP-goat anti-rabbit, rat or mouse) were prepared in Superblock at a ratio of 1:10,000 and added to the plates. The plates were incubated for 1 hour at room temperature. The plates were washed three times with TBS-T. 50 μl/well of POD reagent (prepared according to manufacturer's instructions Sigma-Aldrich and incubated in the dark at room temperature for at least 30 min before use) was added to the plates. After 35 min of incubation protected from light, M5 was used for luminescence reading. The results were plotted in Prism (Fig. 1A-C) using logarithm transformation followed by four-parameter curve fitting. As shown in FIG. 1AC, both AS7B90 and AS7B91 demonstrated specificity for three different FN3 domains, suggesting that the binding epitope of these anti-FN3 domain antibodies is specific to the conserved framework regions rather than the variable loops that provide FN3 domain specificity. It should be noted that no nonspecific binding was detected with the negative control antibody. The mouse version of the AS7B91 antibody appeared to have binding characteristics more similar to the original rabbit hybridoma antibody.

Example 4 Testing AS7B91 to Detect Surface Expressed Anti-BCMA CARTYRIN Primary T Cells.

100 μl per well were seeded with 1x10 anti-BCMA CARTyrin T cells/ml. The wells were washed 2 times with PBS. eFlour 506 Fixable Viability Dye was added to the samples at a final concentration of 1:2000 to stain live-dead cells in the sample and allowed to incubate for 30 min at 4°C. The staining reaction was quenched with FACS buffer, and the samples were washed twice with FACS buffer. Fc Block was added (33 μl Fc/ml FaCS) over 10 min at room temperature. 100 μl of primary AS7B91, FACS buffer, or isotype control were added and incubated for 20 min on ice. The samples were then washed once using FACS buffer. Secondary antibody was added and incubated for 20 min on ice (anti-mouse IgG-AF647 was used at 1:50 for a final concentration of 10 μg/ml). After incubation with the secondary antibody, wells were washed once with FACS buffer and then once with PBS. After washing, samples were fixed in 2% PFA for 10 min at room temperature. Samples were washed and resuspended in FACS buffer.

As shown in FIG. 2 recombinant AS7B91 was able to detect CARTyrin expression on the surface of primary T cells and thus can be used as a general reagent for CARTyrin detection. In a homogeneous population of CARTyrin-expressing T cells, the AS7B91 antibody can be used to quantify CARTyrin on the cell surface.

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Example 5: Use of AS7B91 to activate T cells expressing CARTYRIN.

Because CARTyrins bind to T cell signaling domains, binding and clustering of these CARTyrins by the AS7B91 antibody may result in activation of these signaling domains and activation of the T cells expressing them. To test this, primary T cells expressing CARTyrin were incubated with AS7B91 or an anti-CD3 antibody known to induce activation. Briefly, 12 different RNA clones expressing anti-BCMA CARTyrin were electroporated into primary pan-T cells obtained from healthy human blood (Scripps Normal Blood Donor Service (TSRI)) using an ECM system. 830 Square Wave Electroporation System (BTX). 5X10 ⁶ pan-T cells received a single electrical pulse (500 V, 750 μs) according to the manufacturer's protocol, with or without 10 μg of BCMA-targeting CAR mRNA. Surface expression of CAR was assessed after 24 h using a polyclonal Ab against the FN3 domain. AS7B91 or anti-CD3 antibody was added at a concentration of 5 μg/ml in the presence of soluble anti-CD28 antibody (2 μg/ml). Cells and supernatants were collected on days 2 and 6 after stimulation. Cells were stained for markers of T cell subsets (CD4, 8), activation markers (CD25, 69, 71, 137, HLA-DR) and Fixable Viability Dye. Cells were gated for viability (FVD negative) -> paired cells excluded -> CD4 or CD8 single positive -> CD4 and CD8 were assessed for activation marker expression individually. LSR voltages were established based on antibody-conjugated beads and single-color controls. Gates were set based on fluorescence with a combination of detectable labels without one (FMO) and isotopic controls.

As can be seen from table. 11, the anti-FN3 domain monoclonal antibody AS7B91 is able to stimulate CARTyrin+ primary pan-T cells when co-stimulated via CD28 (similar to procedures with anti-CD3/CD28 antibodies). This activation is dependent on CARTyrin expression and is not as robust or long-lasting as with anti-CD3 antibody. In addition, treatment with AS7B91 and anti-CD28 antibody resulted in an 11-fold increase in cell number compared to unstimulated cells. Thus, an antibody against the FN3 domain can induce T cell proliferation and activation of cells expressing CARTyrin.

Table 11. Activation of pan-T cells expressing CARTyrin using anti-centyrin antibody AS7B91 in combination with co-stimulation with anti-CD28 antibody. Compared with the expression of unstimulated control cells, positive. = positive staining (negative in unstimulated), high = high level of staining, low = low level of staining (negative in unstimulated), negative = no staining.

Table 11

Day 2 CD8 CD25 D.R. CD71 CD69 CD137 positive high high bottom. neg. a-centirin positive high high positive positive a-CD3

Example 6 Construction of the AS7B91 B SCFV chimeric antigen receptor.

To generate the CAR antibody construct AS7B91, AS7B16 and AS7B82, the variable regions were engineered into scFv. For AS7B91, scFv was generated in two different orientations: HCv-LCv, or LCv-HCv. AS7B91 H-L scFv (SEQ ID NO: 22)

METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGIDLSTSVMGW VRQAPGKGLESIGFIYTNVNTYYASWAKGRFTISRTSTTVDLKITSPTTGDTATYFCARA VYAGAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQTPASVSGPVGG

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TVTIKCQASERIYSNLAWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTIRDL

ECADAATYSCQYTSYGSGYVGTFGGGTEVVVEG

AS7B91 L-H scFv (SEQ ID NO: 23)

MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVSGPVGGTVTIKCQASERIYSN

LAWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTIRDLECADAATYSCQYTS YGSGYVGTFGGGTEVVVEGGGGGSGGGGSGGGGSGGGGSLEESGGRLVTPGTPLTLTC TVSGIDLSTSVMGWVRQAPGKGLESIGFIYTNVNTYYASWAKGRFTISRTSTTVDLKITS PTTGDTATYFCARAVYAGAMDLWG QGTLVTVSS

AS7B16 H-L scFv (SEQ ID NO: 66)

QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTGVGWIRQPSGKGLEWLAHIWW

DDDKGYNPALKSRLTISKNTSSNLVFLKIASVDTADTATYYCVRIKGRMDYWGQGTSV TVSSGGGGSGGGGSGGGGSGGGGSNIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGSK QKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISNVQAEDLAVYY CHQYLSLFTFGSGTKLEIK

AS7B16 L-H scFv (SEQ ID NO: 82)

NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGSKQKNYLAWYQQKPGQSPKLLI

YWASTRESGVPDRFTGSGSGSGSGSGTDFILTISNVQADLAVYYCHQYCHQYLSLSLSLSGSGSGGGGGGGGGGSGGSGSGPSLTCSLNTSGTGVGWIRQP SGKGLAWLAH IWWDDDKGYNPALKSRLTISKNLVFLKISVDTADATATYCVRIK GRMDYWGQGTSVTVSS

AS7B82 H-L scFv (SEQ ID NO: 67)

QEQQKESGGGLVKPGASLTLTCTASGIDFSSVAYMCWVRQAPGKGLEWIACIYA

GSSSSIYYASWAKGRFTVSRTSSTTVTLQMTSLTAADTATYFCARGLFTSGSGYYIDMW GPGTLVTVSSGGGGGSGGGGSGGGGSGGGGSDVVMTQTPSSVEVAVGGTVTIKCQASQ SIGSNLAWYQQKPGQRPKLLIYGASNLAAGVPSRFSGSGSGTQFTLTISDVECADAATYY CQRGYISSAVDFFVFGGGTEVVV KG

AS7B82 L-H scFv (SEQ ID NO: 83)

DVVMTQTPSSVEVAVGGTVTIKCQASQSIGSNLAWYQQKPGQRPKLLIYGASNL

AAGVPSRFSGSGSGTQFTLTISDVECADAATYYCQRGYISSAVDFFVFGGGTEVVVKGG GGGGSGGGGSGGGGSGGGGSQEQQKESGGGLVKPGASLTLTCTASGIDFSSVAYMCW VRQAPGKGLEWIACIYAGSSSSIYYASWAKGRFTVSRTSSTTVTLQMTSLTAADTATYF CARGLFTSGSGYYIDMWGPGTLV TVSS

The amino acid sequence of the various scFv sequences was reverse translated and engineered using the hinge sequence, transmembrane domain, and signaling domains. The finished construct was cloned into the T7 transcription vector in vitro to generate mRNA using the commercially available mMESSAGE mMACHINE® T7 ULTRA transcription kit. The amino acid sequences for the components of the CAR constructs were shown in Table. 12.

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Table 12

Amino acid sequence of two different CAR constructs AS7B91, AS7B16 and AS7B82 (H-L orientation and L-H orientation)

Domain Subsequence Extracellular AS7B91 SEQ GO NO: 72 (H-L orientation) QSLEESGGRLVTPGTPLTLTCTVSGIDLSTSVMGWVRQAPGKG LESIGFIYTNVNTYYASWAKGRFTISRTSTTVDLKITSPTTGDTA TYFCARAVYAGAMDLWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSDVVMTQTPASVSGPVGGTVTIKCQASERIYSNLAWYQ QKPGQPPKLLIYKASTLASGVSSRFKGS GSGTEFTLTIRDLECA DAATYSCQYTSYGSGYVGTFGGGTEVVVEG SEQ GO NO: 73 (L-H orientation) DVVMTQTPASVSGPVGGTVTIKCQASERIYSNLAWYQQKPGQ PPKLLIYKASTLASGVSSRFKGSGSGTEFTLTIRDLECADAATY SCQYTSYGSGYVGTFGGGTEVVVEGGGGGSGGGGSGGGGSG GGGSLEESGGRLVTPGTPLTLTCTVSGIDLSTSVMGWVRQAPG KGLESIGFIYTNVNTYYASWAKGRFTIS RTSTTVDLKITSPTTG DTATYFCARAVYAGAMDLWGQGTLVTVSS Extracellular AS7B16 SEQ GO NO: 68 (H-L orientation) QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTGVGWIRQPSG KGLEWLAHIWWDDDKGYNPALKSRLTISKNTSSNLVFLKIASV DTADTATYYCVRIKGRMDYWGQGTSVTVSSGGGGSGGGGSG GGGSGGGGSNIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGS KQKNYLAWYQQKPGQSPKLLIYW ASTRESGVPDRFTGSGSGT DFILTISNVQAEDLAVYYCHQYLSLFTFGSGTKLEIKTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNL GRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR SEQ GO NO: 69 (L-H orientation) NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGSKQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISNVQA EDLAVYYCHQYLSLFTFGSGTKLEIKGGGGSGGGGSGGGGSG GGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTGVGWIR QPSGKGLEWLAHIWWDDDKGYNP ALKSRLTISKNTSSNLVFL KIASVDTADTATYYCVRIKGRMDYWGQGTSVTVSSTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELN LGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR Extracellular AS7B82 SEQ GO NO: 70 (H-L orientation) QEQQKES GGGLVKPG AS LTLTCT AS GIDFS S V A YMC W VRQ AR GKGLEWIACIYAGSSSSIYYASWAKGRFTVSRTSSTTVTLQMT SLTAADTATYFCARGLFTSGSGYYIDMWGPGTLVTVSSGGGG GSGGGGSGGGGSGGGGSDVVMTQTPSSVEVAVGGTVTIKCQA SQSIGSNLAWYQQKPGQRPKLL IYGASNLAAGVPSRFSGSGSG TQFTLTISDVECADAATYYCQRGYISSAVDFFVFGGGTEVVVK GTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQN

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QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR SEQ ID NO: 71 (L-H orientation) DVVMTQTPSSVEVAVGGTVTIKCQASQSIGSNLAWYQQKPGQ RPKLLIYGASNLAAGVPSRFSGSGSGTQFTLTISDVECADAATY YCQRGYISSAVDFFVFGGGTEVVVKGGGGGGSGGGGSGGGGS GGGGSEQQKESGGGLVKPGASLTLTCTASGIDFSSVAYMCWV RQAPGKGLEWIACIYAGSSSSIY YASWAKGRFTVSRTSSTTVTL QMTSLTAADTATYFCARGLFTSGSGYYIDMWGPGTLVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY NELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR human CD8 hinge SEQ GO NO: 24 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIY human transmembrane domain CD8 SEQ ID NO: 25 IWAPLAGTCGVLLLSLVITLYCK intracellular domain of human 41BB SEQ ID NO: 26 RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL intracellular domain of human CD3-zeta SEQ ID NO: 27 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 7 Generation and Analysis of Engineered Immune Cells Expressing the CARs AS7B91, AS7B16, and AS7B82.

The mRNA was electroporated into pan-T cells obtained from healthy human blood (Scripps Blood Services Center) using an ECM 830 Square Wave Electroporation System (BTX). 5X10 ⁶ pan-T cells received a single electrical pulse (500 V, 750 μs) according to the manufacturer's protocol, either with or without 10 μg of CAR AS7B91 mRNA. Surface expression of CAR was assessed after 24 h using a polyclonal Ab against the FN3 domain. The results are presented in Fig. 3.

The functional properties of AS7B91 scFv CAR cells were tested for their ability to bind to FN3 domains and induce T cell killing in response to FN3 domain binding to target cells. This was first determined in a degranulation assay using BCMA-specific FN3 domains together with BCMA ^high (H929 cells), BCMA ^low (D0HH-2 cells) BCMA ^negative (EXPI293 cells) target cells, and then in a killing assay.

In the degranulation assay (CD107a mobilization), T cells were incubated in 96-well plates with cells expressing or not expressing BCMA protein at a ratio of 1:1 or 1:10. Co-cultures were maintained in a final volume of complete OpTmizer medium containing 1:1500 Golgi Stop (BD) and 1:1300 anti-CD107a APC (Biolegend) for 4 h at 37°C. After a 4-hour incubation period, cells were stained with Fixable Vability Dye (eFluor 780 from eBciosience) and anti-CD8 fluorochrome conjugated (PE-conjugated, Biologend) and analyzed by flow cytometry. Degranulation activity was determined by determining the mean fluorescence intensity (MFI) signal for CD107a staining among CD8+ cells. Degranulation assays were performed 24 h after mRNA transfection. As can be seen in FIG. 4, after addition of the anti-BCMA-specific FN3 domain, T cells expressing the AS7B91 scFv CAR were degranulated in an anti-BCMA and AS7B91-specific manner. These data demonstrate that the AS7B91 scFv CAR is functional in its ability to bind and signal in response to a target-specific FN3 domain in which the target is expressed on the surface of other cells.

AS7B91 scFv CAR-T cell killing in response to BCMA-specific FN3 domains along with CFSE-tagged BCMA ^high (U-2932 cells), BCMA ^low (D0HH-2 cells) BCMA ^negative (EXPI293 cells). CAR-T/control (mock-transfected) T cells were preincubated with BCMA-specific FN3 domains for 1 h at 37°C prior to 48 h incubation with BCMA target cells at E:T ratios ranging from 0 to 1. B At the end of the experiment, dead cells were labeled with CFSE-FL1, SYTOX-red-FL4. The percentage of cell death was analyzed using a flow cytometer (FACSCalibur) and the data was analyzed using FlowJo v 10. As seen in FIG. 5, after addition of the BCMA-specific FN3 domain, T cells expressing the AS7B91 scFv CAR killed target cells expressing BCMA with the FN3 domain-specific anti-BCMA and

AS7B91 scFv CAR. These data demonstrate that the AS7B91 scFv CAR is functional in its ability to bind and signal in response to a target-specific FN3 domain in which the target is expressed on the surface of other cells.

For AS7B16 and AS7B82, binding was assessed using flow cytometry. Briefly, 24 h after electroporation, cells were centrifuged at 100× g for 10 min and washed twice in FACS staining buffer (BD Biosciences catalog no. 554657). Cells were incubated with APC-tagged anti-Tencon-25 or AF647-tagged anti-EGFR FN3 83v2 domains at a final concentration of 50 nM at 4°C for 1 h. Labeled cells were washed twice in FACS staining buffer and resuspended in 200 μl of same buffer. Data were collected using a BD LSRFortessa flow cytometer and analyzed using Flowjo software. The data is shown in Fig. 6-fig. 9. All tested CARTs AS7B16, AS7B82 and AS7B91 bind to both FN3 domains tested, Tencon-T25 and EGFR 83v2. CART binding occurs as follows: ASS7B91>AS7B82>AS7B16.

Example 8: In vitro killing of cells expressing anti-citrullinated protein antibody mediated by AS7B91 SCFV CAR T cells pre-complexed with a cyclic citrullinated peptide-conjugated FN3 domain.

Autoimmune diseases are characterized by impaired production of antibodies to autoantigens (autoantibodies). These autoantibodies can be directed against a variety of molecules, including, but not limited to, proteins and nucleic acids. In the case of proteins, these autoantibodies often recognize post-translationally modified antigens. Shown below is the in vitro killing of anti-citrullinated protein antibody (ACPA)-expressing cells mediated by AS7B91 scFv CAR T cells (BAR-T cells) precomplexed with cyclic citrullinated peptide-conjugated centirin (CCP-1). This finding suggests that self-antigens can be conjugated to centirin Tencon25 to interact with BAR T cells, resulting in targeted elimination of antigen-specific self-reactive B cells.

Generation of cell lines expressing FcgR.

Purified lentiviral expression plasmids encoding human FcgRs (CD16a, CD32, and CD64) were packaged for transfection of 293T cells using the Lenti-Pac HIV Expression Packaging System (GeneCopoeia). At 72 h posttransfection, the supernatant containing lentivirus was collected and used to transduce HEK293 cells. The medium was supplemented with polybrene (final concentration - 8 μg/ml). The next day, media containing polybrene were replaced by RPMI 1640 medium supplemented with 10% fetal bovine serum. 72 h after transduction, cells were harvested and stained for FcgR expression. Cells were then sorted based on FcgR expression (SH800S Cell Sorter Sony Biotechnology). Cells with high expression were cultured and used as target cells for future studies.

Conjugation of citrullinated cyclic peptides to centirin Tencon25.

Glycine-tagged (GGG-) citrullinated cyclic peptide-1 (CCP-1-Cit) and arginine control peptide (CCP-1-Arg) were obtained from Peptides International. Sortase-c5-tagged Tencon25 (Tencon25_sort_v5) was desalted in TBS and concentrated before conjugation. Each peptide was conjugated in a 1:5 ratio (FN3 domain to peptide) via sortase chemistry. The conjugates were purified manually on a Ni-Sepharose (GE) column to remove free sortase and peptide. After purification, the buffer was replaced with PBS and the conjugates were concentrated. The conjugates were quality checked by (a) mass spectrometry (LC-MS) and (b) size exclusion chromatography (Superdex 75) and subjected to sterile filtration.

Detection of binding of anticitrullinated antibodies to centirin-CCP1-peptide conjugates

HEK293 cells expressing FcgR were incubated with 200 μg/ml human anti-citrullinated fibrinogen antibody (clone 1F11-Modiquest) or human IgG1 isotype control (Abcam) for 30 min on ice. Cells were washed twice with FACS buffer and then incubated with centirin-CCP1-Cit conjugate (576 nM) for 1 h on ice. Cells were washed twice with FACS buffer and then incubated with PE (phycoerythrin)-labeled anti-centyrin antibody (AS7B91) for 30 min on ice. Binding was assessed by flow cytometry (BD FACSCanto II) and analyzed using BDFACSDiva 6.1.3 software.

AS7B91 scFv CAR T cell-mediated killing of cells expressing FcR associated with an anticitrullinated mAb.

Mock-electroporated T cells (control) or scFv CAR AS7B91 T cells (BAR-T) were precomplexed with centirin-conjugated CCP1 (cit-CCP). HEK293 cells expressing CD16a or CD64 were labeled with Cell Tracker Green (CTG) dye (ThermoFisher) and prebound to either a human IgG1 isotype control or anti-citrullinated fibrinogen antibody (anti-CCP Ab). BAR-T and HEK293 cells were then plated at a ratio of 1:1 or 5:1 for ~18 h. At the end of the experiment, cells were stained using Zom dye

- 36 044007 bie Dye Violet (Biolegend). Dead cells were considered as CTG+/Zombie+ cells as assessed by flow cytometry (BD FACSCanto II). Data were analyzed using BD FACSDiva 6.1.3 and Graphpad Prism 5 software. A 2-fold increase in anticitrullinated antibody-bound target cell death was observed when incubated with BAR-T cells precomplexed with centirin-CCP-1-cit. compared with incubation with control T cells or isotype control antibodies.

Evaluation of potential peptide/antibody combinations in multiple autoimmune diseases to expand the BAR-T platform.

Peptides specific for a) myasthenia gravis (MG), b) multiple sclerosis (MS) and c) systemic lupus erythematosus (SLE) were tested for their ability to bind to anti-AChR, anti-MOG or anti-dsDNA antibodies, respectively. Three high-affinity neutravidin plates were coated with 10 μg/mL of the indicated peptides. Plates were washed 3 times with PBS-T and blocked with 2X Assay Buffer A (eBioscience) for 2 h at room temperature. The plates were washed 3 times with PBS-T and 100 μg/ml of the indicated antibody was added for 1 h at room temperature. The plates were washed 3 times with PBS-T and the appropriate secondary antibody was added for 1 h at room temperature. The plates were washed 3 times with PBS-T and TMB substrate was added for 5-8 min before adding Stop Solution (ThermoFisher). A SpectraMax340PC instrument was used for absorbance readings, and data were analyzed using Graphpad Prism 5 software. Binding results identified one potential peptide/antibody combination for each disease indication for further evaluation.

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Brief description of the sequence list.

SEQ ID NO: Type View Description Subsequence 1 PRT Rabbit HCDR1 CEN25-105-5 IMGT GIDLSTSV 2 PRT Rabbit HCDR1 CEN25-105-5 Kabatu TSVMG 3 PRT Rabbit HCDR1 CEN25-105-5 Chotia GIDLSTS 4 PRT Rabbit HCDR2 CEN25-105-5 IMGT IYTNVNT 5 PRT Rabbit HCDR2 CEN25-105-5 Kabatu FIYTNVNTYYASWAKG 6 PRT Rabbit HCDR2 CEN25-105-5 Chotia YTNVN 7 PRT Rabbit HCDR3 CEN25-105-5 IMGT ARAVYAGAMDL 8 PRT Rabbit HCDR3 CEN25-105-5 Kabatu and Chotia AVYAGAMDL 9 PRT Rabbit LCDR1 CEN25-105-5 IMGT ERIYSN 10 PRT Rabbit LCDR1 CEN25-105-5 Kabatu and Chotia QASERIYSNLA eleven PRT Rabbit LCDR2 CEN25-105-5 IMGT KAS 12 PRT Rabbit LCDR2 CEN25-105-5 Kabatu and Chotia KASTLAS 13 PRT Rabbit LCDR3 CEN25-105-5 IMGT, Kabatu and Chotia QYTSYGSGYVGT 14 PRT Rabbit VH CEN-25105-5 QSLEESGGRLVTPGTPLTLTCTVSGIDLSTSVMG WVRQAPGKGLESIGFIYTNVNTYYASWAKGRF TISRTSTTVDLKITSPTTGDTATYFCARAVYAGA

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MDLWGQGTLVTVSS 15 PRT Rabbit VL CEN-25105-5 DVVMTQTPASVSGPVGGTVTIKCQASERIYSNL AWYQQKPGQPPKLLIYKASTLASGVSSRFKGSG SGTEFTLTIRDLECADAATYSCQYTSYGSGYVG TFGGGTEVVVEG 16 DN to Rabbit VH CEN-25105-5 Ctggagggagtccgggggtcgcctggtcacgcctgggacacccctgacac tcacctgcacagtctctggaatcgacctcagtacctctgtcatgggttgggtc cgccaggctccaggaaggggctggaatccatcggattcatttatactaat gttaacacatactacgcgagctgggcaaaaggccgattcaccatctcca ga acctcgaccacggtggatctgaaaatcaccagtccgacaaccggggacac ggccacctatttctgtgccagagctgtttatgctggtgctatggacttgtggg gccaaggcaccctggtcaccgtctcctca 17 DN to Rabbit VL CEN-25105-5 gatgttgtgatgacccagactccagcctccgtgtctggacctgtgggaggc acagtcaccatcaagtgccaggccagtgagagaatttatagcaatttagcct ggtatcagcagaaaccagggcagcctcccaaactcctgatctacaaggca tccactctggcatctggggtctcatcgcggttcaaaggcagtggatctggga cagag ttcactctcaccatcagggaccttgagtgtgccgatgctgccactta ctcctgtcaatatacttcttatggcagtggttatgttggtactttcggcggagg gaccgaggtggtggtcgaaggt 18 PRT Artificial naya Heavy chain AS7B91 HP ASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLG GPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDP DVQISWFVNNVEVHTAQTQTHREDYNSTLRVV SALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYK NTEPVLDS DGSYFMYSKLRVEKKNWVERNSYSCSVVHEGL HNHHTTKSFSRTPGK 19 PRT Artificial naya Light chain AS7B91 DVVMTQTPASVSGPVGGTVTIKCQASERIYSNL AWYQQKPGQPPKLLIYKASTLASGVSSRFKGSG SGTEFTLTIRDLECADAATYSCQYTSYGSGYVG TFGGGTEVVVEGRADAAPTVSIFPPSSEQLTSGG ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCE ATHKTSSTSPIVKSFNRNEC 20 PRT Artificial naya Heavy chain AS7B90 QSLEESGGRLVTPGTPLTLTCTVSGIDLSTSVMG WVRQAPGKGLESIGFIYTNVNTYYASWAKGRF TISRTSTTVDLKITSPTTGDTATYFCARAVYAGA MDLWGQGTLVTVSSAETTAPSVYPLAPGTALK SNSMVTLGCLVKGYFPEPVTVTWNSGALSSGV HTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCN VAHPASSTKVDKKIVPRNCGGDCKPCICTGSEV SSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPE VHFSWFVDDVEVHTAQTRPPEEQFNSTFRSVSE LPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPE GRTQVPHVYTMSPTKEEMTQNEVSITCMVKGF YPPDIYVEWQMNGQPQENYK NTPPTMDTDGSY

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FLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHH TEKSLSHSPGK 21 PRT Artificial naya Light chain AS7B90 DVVMTQTPASVSGPVGGTVTIKCQASERIYSNL AWYQQKPGQPPKLLIYKASTLASGVSSRFKGSG SGTEFTLTIRDLECADAATYSCQYTSYGSGYVG TFGGGTEVVVEGRADAAPTVSIFPPSSEQLASGG ASVVCFINKFYPKDISVKWKIDGSERQNDVLNS VTDQDSKDSTYSMSSTLTLTKADY ERHNLYTCE VVHKTSASPVVKSFNRNEC 22 PRT Artificial naya AS7B91 H-L scFv METGLRWLLLVAVLKGVQCQSLEESGGRLVTP GTPLTLTCTVSGIDLSTSVMGWVRQAPGKGLES IGFIYTNVNTYYASWAKGRFTISRTSTTVDLKIT SPTTGDTATYFCARAVYAGAMDLWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSDVVMTQTPA SVSGPVGGTVTIKCQASERIYSNLAWYQQKPGQ PPK LLIYKASTLASGVSSRFKGSGSGTEFTLTIRD LECADAATYSCQYTSYGSGYVGTFGGGTEVVV EG 23 PRT Artificial naya AS7B91 L-H scFv MDTRAPTQLLGLLLLWLPGARCDVVMTQTPAS VSGPVGGTVTIKCQASERIYSNLAWYQQKPGQP PKLLIYKASTLASGVSSRFKGSGSGTEFTLTIRDL ECADAATYSCQYTSYGSGYVGTFGGGTEVVVE GGGGGSGGGGSGGGGSGGGGSLEESGGRLVTP GTPLTLTCTVSGIDLSTSVMGWVRQAPGKGLES IGFIY TNVNTYYASWAKGRFTISRTSTTVDLKIT SPTTGDTATYFCARAVYAGAMDLWGQGTLVT VSS 24 PRT Human CD8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIY 25 PRT Human CD8 transmembrane domain IWAPLAGTCGVLLLSLVITLYCK 26 PRT Human Intracellular domain 4-1BB RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCEL 27 PRT Human Intracellular domain CD3zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 28 PRT Artificial naya Tepsop25 LPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVKGGHRSNPLSAIFTT 29 PRT Artificial naya Tepsop28 MLPAPKNLVVSEVTHDSARLSWTAPDAAFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKHHTE YTVSIYGVKGGHRSNPLSAIFHTHHHHHH thirty PRT Artificial naya R114-83 MLPAPKNLVVSRVTHDSARLSWTAPDAAFDSF WIRYFEFLGSGEAIVLTVPGSERSYDLTGLKHHT EYVVNIMGVKGGKISPPLSAIFTTHHHHHH 31 PRT Artificial naya AZ MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSF WIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGT EYVVNIMGVKGGKISPPLSAIFTTGGHHHHHH 32 PRT Artificial naya Sequence 83v2-ABD MLPAPKNLVVSEVTEDSARLSWDDPWAFYESF LIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGT EYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGG

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SGGGGSLAEAKVLANRELDKYGVSDYYKNLIN NAKTVEGVKALIDEILAALPGGHHHHHH 33 PRT Artificial naya Tepsop LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEY TVSIYGVKGGHRSNPLSAEFTT 34 PRT Artificial naya Tepsop27 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLI QYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVKGGHRSNPLSAIFTT 35 PRT Mouse HCDR1 AS7B16 by IMGT GFSLNTSGTG 36 PRT Mouse HCDR1 AS7B16 by Kabat TSGTGVG 37 PRT Mouse HCDR1 AS7B16 by Chotia GFSLNTSGT 38 PRT Rabbit HCDR1 AS7B82 by IMGT GIDFSSVAY 39 PRT Rabbit HCDR1 AS7B82 by Kabat SVAYMC 40 PRT Rabbit HCDR1 AS7B82 by Chotia GIDFSSVA 41 PRT Mouse HCDR2 AS7B16 by IMGT IWWDDDK 42 PRT Mouse HCDR2 AS7B16 by Kabat HIWWDDDKGYNPALKS 43 PRT Mouse HCDR2 AS7B16 by Chotia WWDDD 44 PRT Mouse HCDR3 AS7B16 by IMGT VRIKGRMDY 45 PRT Mouse HCDR3 AS7B16 by Kabat and Chotia IKGRMDY 46 PRT Mouse LCDR1 AS7B16 by IMGT QSVLFGSKQKNY 47 PRT Mouse LCDR1 AS7B16 by Kabat and Chotia KSSQSVLFGSKQKNYLA 48 PRT Mouse LCDR2 AS7B16 by IMGT W.A.S. 49 PRT Mouse LCDR2 AS7B16 by Kabat and Chotia WASTRES 50 PRT Mouse LCDR2 AS7B16 according to IMGT, Kabatu and Chotia HQYLSLFT 51 PRT Rabbit HCDR2 AS7B82 by IMGT IYAGSSSSI 52 PRT Rabbit HCDR2 AS7B82 by Kabat CIYAGSSSSSIYYASWAKG 53 PRT Rabbit HCDR2 AS7B82 by Chotia YAGSSSS 54 PRT Rabbit HCDR3 AS7B82 ARGLFTSGSGYYIDM

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by IMGT 55 PRT Rabbit HCDR3 AS7B82 by Kabat and Chotia GLFTSGSGYYIDM 56 PRT Rabbit LCDR1 AS7B82 by IMGT QSIGSD 57 PRT Rabbit LCDR1 AS7B82 by Kabat and Chotia QASQSIGSNLA 58 PRT Rabbit LCDR2 AS7B82 by IMGT SAS 59 PRT Rabbit LCDR2 AS7B82 by Kabat and Chotia GASNLAA 60 PRT Rabbit LCDR3 AS7B82 by IMGT QCTYSSSTGYNA 61 PRT Rabbit LCDR3 AS7B82 by Kabat and Chotia QRGYISSAVDFFV 62 PRT Artificial naya Heavy chain AS7B16 N VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEV QFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDF FPEDITVEWQWNGQPAEN YKNTQPIMNTNGSY FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHH TEKSLSHSPGK 63 PRT Artificial naya Light chain AS7B16 NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGS KQKNYLAWYQQKPGQSPKLLIYWASTRESGVP DRFTGSGSGTDFILTISNVQAEDLAVYYCHQYLS LFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGG ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCE ATHKTSSTSPIVKSFNRNEC 64 PRT Artificial naya Heavy chain AS7B82 LG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGF YPSDIAVEWESNGQPEN

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NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 65 PRT Artificial naya Light chain AS7B82 DVVMTQTPSSVEVAVGGTVTIKCQASQSIGSNL AWYQQKPGQRPKLLIYGASNLAAGVPSRFSGSG SGTQFTLTISDVECADAATYYCQRGYISSAVDFF VFGGGTEVVVKGRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 66 PRT Artificial naya AS7B16 H-L scFv QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTG VGWIRQPSGKGLEWLAHIWWDDDKGYNPALK SRLTISKNTSSNLVFLKIASVDTADTATYYCVRI KGRMDYWGQGTSVTVSSGGGGSGGGGSGGGG SGGGGSNIMMTQSPSSLAVSAGEKVTMNCKSS QSVLFGSKQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFILTISNVQAEDLAVY YCHQYLSLFTFGSGTKLEIK 67 PRT Artificial naya AS7B82 H-L scFv QEQQKESGGGLVKPGASLTLTCTASGIDFSSVA YMCWVRQAPGKGLEWIACIYAGSSSSIYYASW AKGRFTVSRTSSTTVTLQMTSLTAADTATYFCA RGLFTSGSGYYIDMWGPGTLVTVSSGGGGGSG GGGSGGGGSGGGGSDVVMTQTPSSVEVAVGGT VTIKCQASQSIGSNLAWYQQKPGQRPKLLIYGA SNLAAGVPSRFSGSGSGTQFTLTISDVECADAAT YYCQRGYISSAVDFFVFGGGTEVVVKG 68 PRT Artificial naya VKD AS7B16HLCAR QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTG VGWIRQPSGKGLEWLAHIWWDDDKGYNPALK SRLTISKNTSSNLVFLKIASVDTADTATYYCVRI KGRMDYWGQGTSVTVSSGGGGSGGGGSGGGG SGGGGSNIMMTQSPSSLAVSAGEKVTMNCKSS QSVLFGSKQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFILTISNVQAEDLAVY YCHQYLSLFTFGSGTKLEIKTTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK FSRSADAPAYKQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR 69 PRT Artificial naya VKD AS7B16 ΤΗ CAR NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGS KQKNYLAWYQQKPGQSPKLLIYWASTRESGVP DRFTGSGSGTDFILTISNVQAEDLAVYYCHQYLS LFTFGSGTKLEIKGGGGSGGGGSGGGGSGGGGS QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTG VGWIRQPSGKGLEWLAHIWWDDDKGYNP ALK SRLTISKNTSSNLVFLKIASVDTADTATYYCVRI KGRMDYWGQGTSVTVSSTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYKQGQNQLYNELNLGRREEYDV LDK

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RRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR 70 PRT Artificial naya VKD AS7B82 НLCAR QEQQKESGGGLVKPGASLTLTCTASGIDFSSVA YMCWVRQAPGKGLEWIACIYAGSSSSIYYASW AKGRFTVSRTSSTTVTLQMTSLTAADTATYFCA RGLFTSGSGYYIDMWGPGTLVTVSSGGGGGSG GGGSGGGGSGGGGSDVVMTQTPSSVEVAVGGT VTIKCQASQSIGSNLAWYQQKPGQRPKLLIYGA SNLAAGVPSRFSGSGSGTQFTLTISDVECADAAT YYCQRGYISSAVDFFVFGGGTEVVVKGTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYKQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 71 PRT Artificial naya VKD AS7B82 LHCAR DVVMTQTPSSVEVAVGGTVTIKCQASQSIGSNL AWYQQKPGQRPKLLIYGASNLAAGVPSRFSGSG SGTQFTLTISDVECADAATYYCQRGYISSAVDFF VFGGGTEVVVKGGGGGGSGGGGSGGGGSGGG GSEQQKESGGGLVKPGASLTLTCTASGIDFSSVA YMCWVRQAPGKGLEWIACIYAGSSSSIYY ASW AKGRFTVSRTSSTTVTLQMTSLTAADTATYFCA RGLFTSGSGYYIDMWGPGTLVTVSSTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYKQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 72 PRT Artificial naya VKD AS7B91 НLCAR G SGSGTEFTLTIRDLECADAATYSCQYTSYGSG YVGTFGGGTEVVVEG 73 PRT Artificial naya VKD AS7B91 ΤΗ CAR DVVMTQTPASVSGPVGGTVTIKCQASERIYSNL AWYQQKPGQPPKLLIYKASTLASGVSSRFKGSG SGTEFTLTIRDLECADAATYSCQYTSYGSGYVG TFGGGTEVVVEGGGGGSGGGGSGGGGSGGGGS LEESGGRLVTPGTPLTLTCTVSGIDLSTSVMGW VRQAPGKGLESIGFIYTNVNTYYASWAK GRFTI SRTSTTVDLKITSPTTGDTATYFCARAVYAGAM DLWGQGTLVTVSS 74 PRT Artificial naya VH AS7B16 QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGTG VGWIRQPSGKGLEWLAHIWWDDDKGYNPALK SRLTISKNTSSNLVFLKIASVDTADTATYYCVRI

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KGRMDYWGQGTSVTVSS 75 PRT Artificial naya VL AS7B16 NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLFGS KQKNYLAWYQQKPGQSPKLLIYWASTRESGVP DRFTGSGSGTDFILTISNVQAEDLAVYYCHQYLS LFTFGSGTKLEIK 76 DN K Artificial naya VH AS7B16 AGGTTACTCTGAAAGAGTCTGGCCCTGGGATA TTGCAGCCCTCCCAGACCCTCAGTCTGACTTG TTCTTTCTCTGGGTTTTCACTGAACACTTCTGG TACGGGTGTAGGCTGGATTCGTCAGCCTTCAG GGAAGGGTCTGGAGTGGCTGGCACACATTTG GTGGGATGATGACAAGGGGTATAACCCAGCC CTGAAGAGCCGACTGACAATCTCCAAAACA CCTCCAGCAACCT GGTATTCCTCAAGATCGCC AGTGTGGACACTGCAGATACTGCCACATATTA CTGTGTTCGAATCAAAGGCCGGATGGACTACT GGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 77 DN to Artificial naya VL AS7B16 AACATTATGATGACACAGTCGCCATCCCTCTCT GGCTGTGTCTGCAGGAGAAAAGGTCACTATG AACTGTAAGTCCAGTCAAAGTGTTTTATTCGG TTCAAAACAGAAGAACTATTTGGCCTGGTACC AGCAGAAACCAGGGCAGTCTCCTAAATTGCT GATCTACTGGGCATCCACTAGGGAATCTGGTG TCCCTGATCGCTTCACAGGCAGTGGATCTGGG ACAGATTTTATACT TACCATCAGCAATGTACA AGCTGAAGACCTGGCAGTTTATTACTGTCATC AATACCTCTCCCTATTCACGTTCGGCTCGGGG ACAAAGTTGGAAATAAAA 78 PRT Artificial naya VH AS7B82 QEQQKESGGGLVKPGASLTLTCTASGIDFSSVA YMCWVRQAPGKGLEWIACIYAGSSSSIYYASW AKGRFTVSRTSSTTVTLQMTSLTAADTATYFCA RGLFTSGSGYYIDMWGPGTLVTVSS 79 PRT Artificial naya VL AS7B82 DVVMTQTPSSVEVAVGGTVTIKCQASQSIGSNL AWYQQKPGQRPKLLIYGASNLAAGVPSRFSGSG SGTQFTLTISDVECADAATYYCQRGYISSAVDFF VFGGGTEVVVKG 80 DN K Artificial naya VH AS7B16 CAGGAGCAGCAGAAGGAGTCCGGGGGAGGCC TGGTCAAGCCTGGGGCATCCCTGACACTCACC TGCACAGCTTCTGGAATCGACTTCAGTAGTGT CGCCTACATGTGTTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGAGTGGATCGCATGCATTTA TGCTGGTAGTAGTAGTAGCATCTACTACGCGA GCTGGGCGAAAGGCCGATTCACCGTCTCCAG AACCT CGTCTACCACGGTGACTCTGCAAATGA CCAGTCTGACAGCCGCGGACACGGCCACCTA TTTCTGTGCGAGAGGTCTATTTACTAGTGGTA GTGGATATTATATAGACATGTGGGGCCCAGG CACCCTGGTCACCGTCTCCTCA 81 DN to Artificial naya VL AS7B16 GATGTCGTGATGACCCAGACTCCATCCTCTGT GGAGGTAGCTGTGGGAGGCACAGTCACCATC AAGTGCCAGGCCAGTCAGAGCATTGGTAGTA ATTTAGCCTGGTATCAGCAGAAACCAGGGCA GCGTCCCAAGCTCCTGATCTATGGTGCATCCA ATCTGGCCGCTGGGGTCCCATCCGGTTCAGT GGCAGTGGATCTGGGACACAGTTCACTCTCAC CATCAGCGACGTGGAGTGTGCCGATGCTGCC ACTTACTACTGTCAACGGGGTTATATTAGCAG TGCTGTTGATTTTTTTGTTTTCGGCGGAGGGA CCGAGGTGGTGGTCAAAGGT

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Claims (1)

Watanabe et al., J Biol Chern 265:15659-15665, 1990 Zielske et al., J Clin Invest. 2003 Nov;l 12(10):1561-70 US2010/0216708 US2013/0226834 US2011/0274623 US4767704 US4657866 US4927762 US4560655 US5122469 WO90/03430 WO87/00195 USRE30985 WO 2013/176915 WO2014/130635 WO2013/176916 WO2013/176915 WO2014/039523 WO2014/184741 WO2014/191128 WO2014/184744 WO2014/184143 US6352694 US6534055 US6905680 US6692964 US5858358 US6887466 US6905681 US7144575 US7067318 US7172869 US7232566 US7175843 US5883223 US6905874 US6797514 US6867041 US2006/121005 US6040177 US5827642 WO2012129514 CLAIM A chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising a scFv that contains the amino acid sequence of SEQ ID NO: 72 or 73; wherein the extracellular domain specifically binds to a non-randomized region of the FN3 domain; (b) a transmembrane domain which contains the amino acid sequence of SEQ ID NO: 25; (c) an intracellular signaling domain, which contains the amino acid sequence of SEQ ID NO: 26, and a primary signaling domain, which contains the amino acid sequence -