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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70517—CD8
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the field of the invention is the use of chimeric surface membrane proteins for signal transduction.
• the cells expressing such proteins are configured to recognize and act on cells expressing TAG-72.
• the proteins can contain an antigen-binding moiety which recognizes TAG-72.
• Regulation of cell activities is frequently achieved by the binding of the ligand to a surface membrane receptor.
• the formation of the complex with the extracellular portion of the receptor results in a change in conformation with the cytoplasmic portion of the receptor undergoing a change which results in a signal being transduced in the cell.
• the change in the cytoplasmic portion results in binding to other proteins, where the other proteins are activated and may carry out various functions.
• the cytoplasmic portion is autophosphorylated or phosphorylated, resulting in a change in its activity.
• secondary messengers such as calcium, cyclic adenosine monophosphate, inositol phosphate, diacylglycerol, and the like.
• the binding of the ligand results in a particular signal being induced.
• T cells There are a number of instances, where one might wish to have a signal induced by virtue of employing a different ligand. For example, one might wish to activate particular T cells, where the T cells will then be effective as cytotoxic agents, or activating agents by secretion of interleukins, colony stimulating factors or other cytokines, which results in the stimulation of another cell.
• T cell receptor The ability of the T cell receptor to recognize antigen is restricted by the nature of Major Histocompatibility Complex (MHC) antigens on the surface of the host cell.
• MHC Major Histocompatibility Complex
• a chimeric T cell receptor in which a non-MHC restricted ligand binding domain is linked directly to the signal transducing domain of the T cell receptor would permit the use of the resulting engineered effector T cell in any individual, regardless of their MHC genetic background. In this manner, one may change the ligand which initiates the desired response, where for some reason, the natural agent may not be as useful.
• T cell antigen receptor has a non-covalent association between a heterodimer, the antigen/MHC binding subunit ; variable component and the five invariant chains: zeta ( ⁇ ) , eta ( ⁇ ) and the three CD3 chains: gamma ( ⁇ ), delta ( ⁇ ) and epsilon (e) (Weiss & Imboden (1987) Adv. Immunol. 41:1-38; Cleavers et al. (1988) Ann. Rev. Immunol. 6:629-662; Frank et al. (1990) Sem. Immunol. 2:89-97).
• the physically associated CD3-zeta/eta complex does not bind ligand, but is thought to undergo structural alterations as a consequence of T j -antigen interaction which results in activation of intracellular signal transduction mechanisms.
• the zeta( ⁇ ) chain is structurally unrelated to the three CD3 chains, and exists primarily as a disulfide-linked homodimer, or as a heterodimer with an alternatively spliced product of the same gene, eta ( ⁇ ).
• the zeta chain is also expressed on natural killer cells as part of the Fc ⁇ RIII receptor.
• the gamma chain of the Fc ⁇ receptor is closely related to zeta, and is associated with the Fc ⁇ RI receptor of mast cells and basophils and the C16 receptor expressed by macrophages and natural killer cells.
• the three CD3 chains, gamma ( ⁇ ), delta ( ⁇ ) and epsilon (e), are structurally related polypeptides and were originally implicated in signal transduction of T cells by studies in which anti-CD3 monoclonal antibodies were shown to mimic the function of antigen in activating T cells (Goldsmith & Weiss (1987) Proc. Natl. Acad. Sci. USA 84:6879-6883), and from experiments employing somatic cell mutants bearing defects in TCR-mediated signal transduction function (Sussman et al. (1988) Cell 52:85-95). Sequences similar to the active motif found in zeta are also present in the cytoplasmic domains of the CD3 chains gamma and delta.
• Chimeric receptors in which the cytoplasmic domain of an unrelated receptor has been replaced by that of CD3 epsilon have been shown to be proficient in signal transduction (Letourneur & Klausner (1992) Science 255:79-82), and a 22 amino acid sequence in the cytoplasmic tail of CD3 epsilon was identified as the sequence responsible.
• the cytoplasmic domains of both zeta and CD3 epsilon have been shown to be sufficient for signal transduction, quantitatively distinct patterns of tyrosine pho ⁇ phorylation were observed with these two chains, suggesting that they may be involved in similar but distinct biochemical pathways in the cell.
• TCR T cell receptor
• T cells using a chimeric receptor consisting of the extracellular domains of the chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domain of either ⁇ or ⁇ .
• Gross et al. ((1989) Proc. Natl. Acad. Sci. USA 86:10024-10028) describe activation of T cells using chimeric receptors in which the MHC-restricted antigen-binding domains of the T cell receptor ⁇ and ⁇ chains were replaced by the antigen-binding domain of an antibody.
• Romeo & Seed ((1991) Cell 64:1037-1046) describe activation of T cells via chimeric receptors in which the extracellular portion of CD4 is fused to the transmembrane and intracellular portions of ⁇ , ⁇ , and ⁇ subunits.
• Letourner E Klausner (1992) describe activation of T cells by a chimeric receptor consisting of the extracellular domain of the IL-2 receptor and the cytoplasmic tail of CD3 epsilon (Science 255:79-82).
• Ig immunoglobulin
• T alpha ( ⁇ ) and beta ( ⁇ ) T cell receptor chains
• chimeric Ig-T molecules in which the V domains of the Ig heavy (V H ) and light (V L ) chains are combined with the C regions of Tj o and T ; ⁇ chains have been described (Gross et al. (1989) Proc. Natl. Acad. Sci. USA 86:10024-10028).
• the role of the j chains is to bind antigen presented in the context of MHC.
• the j heterodimer does not possess innate signalling capacity, but transmits the antigen binding event to the CD3/zeta chains present in the TCR complex.
• V H -T V H -T
• V L -Tj V H -Tj chimeric molecules.
• the chimeras have been demonstrated to act as functional receptors by their ability to activate T cell effector function in response to cross-linking by the appropriate hapten or anti-idiotypic antibody (Becker et al. (1989) Cell 58:911 and Gross et al. (1989) Proc. Natl. Acad. Sci. USA 86:10024).
• the V H -T j and V L -T j chains do not possess innate signalling capacity, but act via the CD3/zeta complex.
• TIL's Autologous tumor-infiltrating lymphocytes
• T cell adoptive immunotherapies A significant drawback of all of those T cell adoptive immunotherapies is the prolonged culture time necessary to generate antigen-specific therapeutically relevant numbers of cells.
• An alternative approach is the genetic modification of patient T cells to express a chimeric receptor conferring the ability of MHC independent lysis of the target cell. HLA-unrestricted chimeric T cell receptors can redirect the antigenic-specificity of T cell populations to recognize antigens of choice. On binding to tumor antigen, the chimeric receptors can initiate T cell activation, resulting in induction of effector functions including cytolysis of the tumor cell.
• TAG-72 is a human oncofetal, pancarcinoma sialylated mucin antigen originally described by Schlom et al. at the NCI. TAG-72 is expressed on a variety of tumors including colon, breast, prostate, non-small cell lung and ovarian carcinomas (Thor et al. (1986) Cancer Res. 46:3118). Normal tissue reactivity is limited to low level expression in transitional colonic epithelium and secretory endometrium (Thor et al. (1986) supra).
• TAG-72 expression is heterogeneous
• data from clinical trials conducted at several centers demonstrated that TAG-72 expression on colon, ovarian and breast carcinomas is up-regulated with intraperitoneal (ip) administration of ⁇ -interferon (IFN- ⁇ ) (Greiner et al. (1992) J. Clin. Oncol.) or intravenous (iv) administration of ⁇ -interferon (IFN- ⁇ ) (Roselli et al. (1996) J. Clin. Oncol. ) .
• IFN- ⁇ ⁇ -interferon
• IFN- ⁇ intravenous
• the murine monoclonal antibody cc49 is one of several generated against human TAG-72 that recognize bound protein lysates of human carcinomas but not normal tissues (reviewed in: Schlom et al. in Serological Cancer Markers, Sell, ed. , Humana Press, 1992, Totowa, NJ) .
• Over 400 patients with colorectal, breast, prostate or ovarian carcinomas have been infused with radiolabelled murine cc49 mAb in radioimaging or radioimmunotherapy protocols without reported significant adverse side effects.
• cc49 single chain antibody scFv or scAb
• scFv or scAb mouse-human chimeric cc49
• humanized cc49 antibody Kashmiri et al. (1995) Hybridoma 14:461-473
• humanized cc49 scFV humanized cc49 scFV
• mouse-human chimeric cc49 has a similar TAG-72 binding affinity, whereas the cc49 scFv has an 8-fold lower and the humanized cc49 antibody a 2-fold to 3-fold lower binding affinity for TAG-72.
• In vivo tumor targeting in a mouse xenograft model was equivalent for humanized and chimeric cc49 antibodies (Kashmiri et al. (1995) Hybridoma 14:461-473).
• the triggering of signal transduction leading to cytotoxic function by different cells of the immune system can be initiated by chimeric receptors with antibody-type specificity.
• the chimeric receptors by-pass the requirement for matching at the MHC locus between target cell (i.e. virally-infected, tumor cell, etc.) and effector cell (i.e., T cell, granulocyte, mast cell etc.).
• Intracellular signal transduction or cellular activation is achieved by employing chimeric proteins having a cytoplasmic region associated with transduction of a signal and activation of a secondary messenger system, frequently involving a kinase, and a non-MHC restricted extracellular region capable of binding to a specific ligand and transmitting to the cytoplasmic region the formation of a binding complex.
• cytoplasmic sequences of the zeta, eta, delta, gamma and epsilon chains of TCR and the gamma chain of Fc.RI, or a tyrosine kinase are employed joined to other than the natural extracellular region by a transmembrane domain.
• the extracellular domain can comprise a portion or derivative of an antibody which binds TAG-72 and retains the TAG-72 specificity.
• the antibody can be polyclonal or monoclonal.
• a preferred derivative is a single chain antibody which binds TAG-72.
• Figure 1 is a diagrammatic depiction of the structure of single-chain antibodies used in the chimeric receptors of the invention as compared to the structure of native monoclonal antibodies.
• Figure 2 depicts a map of a chimera comprising a single-chain cc49 extracellular portion and zeta as the intracellular portion of the molecule.
• Figure 3 depicts results of chromium release assays using cells armed with a cc49-zeta chimera.
• Figure 4 depicts the results of chromium release assays using various cancer cell lines as targets.
• Figure 5 depicts the result of a chromium release assay using a mixed population of target cells comprising TAG-72 + and TAG-72" cells.
• Figure 6 depicts the results of a chromium release assay using CD4 cells as effectors.
• Novel DNA sequences such as DNA sequences as parts of expression cassettes and vectors, as well as their presence in cells are provided, where the novel sequences comprise three domains which do not naturally exist together: (1) a cytoplasmic domain, which normally transduces a signal resulting in activation of a messenger system, (2) a transmembrane domain, which crosses the outer cellular membrane, and (3) a non-MHC restricted extracellular receptor domain which serves to bind to a ligand and transmit a signal to the cytoplasmic domain, resulting in activation of the messenger system.
• a preferred extracellular domain is an antibody or antigen-binding .portion thereof, particularly one that binds TAG-72.
• the cytoplasmic domain may be derived from a protein which is known to activate various messenger systems, normally excluding the G proteins.
• the protein from which the cytoplasmic domain is derived need not have ligand binding capability by itself, it being sufficient that such protein may associate with another protein providing such capability.
• Cytoplasmic regions of interest include the zeta chain of the T cell receptor, the eta chain, which differs from the zeta chain only in its most C-terminal exon as a result of alternative splicing of the zeta mRNA, the delta, gamma and epsilon chains of the T cell receptor (CD3 chains) and the gamma subunit of the Fc,RI receptor, and such other cytoplasmic regions which are capable of transmitting a signal as a result of interacting with other proteins capable of binding to a ligand.
• cytoplasmic regions or functional fragments or mutants thereof may be employed, generally ranging from about 10 to 500 amino acids, where the entire naturally occurring cytoplasmic region may be employed or only an active portion thereof.
• the cytoplasmic regions of particular interest are those which may be involved with one or more secondary messenger pathways, particular pathways involved with a protein kinase, more particularly, protein kinase C (PKC) .
• PKC protein kinase C
• Pathways of interest include the phosphatidylinositol-specific phospholipase involved pathway, which is normally involved with hydrolysis of phosphatidylinositol-4, 5-bisphosphate, which results in production of the secondary messengers inositol-1,4, 5-trisphosphate and diacylglycerol.
• Another pathway is the calcium mediated pathway, which may be as a result of direct or indirect activation by the cytoplasmic portion of the chimeric protein.
• the kinase pathway may be involved, which may involve phosphorylation of the cytoplasmic portion of the chimeric protein.
• One or more amino acid side chains, particularly tyrosines, may be phosphorylated.
• fyn a member of the src family of tyrosine kinases
• zeta chain may be involved with the zeta chain. While usually the entire cytoplasmic region will be employed, in many cases, it will not be necessary to use the entire chain. To the extent that a truncated portion may find use, such truncated portion may be used in place of the intact chain.
• Suitable cytoplasmic domains arise also from other molecules that have a signalling role in eliciting a response by the host cell.
• tyrosine kinases such as ZAP-70 and members of the Janus kinase family
• ancillary molecules that have less than a direct role in signaling, such as CD2 and CD28, or functional portions thereof, can be found as the cytoplasmic domain of a receptor of interest. See W096/23814.
• a desirable response by the host cell is proliferation or differentiation. Manifestation of a desirable phenotype, such as cytotoxicity, is obtained and which can be directed to a specific target, such as a cancer cell, by use of a chimeric receptor of interest.
• the transmembrane domain may be the domain of the protein contributing the cytoplasmic portion, the domain of the protein contributing the extracellular portion, or a domain associated with a totally different protein.
• Chimeric receptors of the invention in which the transmembrane domain is replaced with that of a related receptor, or, replaced with that of an unrelated receptor, may exhibit qualitative and/or quantitative differences in signal transduction function from receptors in which the transmembrane domain is retained.
• functional differences in signal transduction may be dependent not only upon the origin of the cytoplasmic domain employed, but also on the derivation of the transmembrane domain.
• transmembrane domain naturally associated with one or the other of the other domains, particularly the extracellular domain.
• transmembrane domain of the zeta, eta, or Fc.RI gamma chains which contain a cysteine residue capable of disulphide bonding so that the resulting chimeric protein will be able to form disulfide-linked dimers with itself, or with unmodified versions of the zeta, eta, or FceRI gamma chains or related proteins.
• the transmembrane domain will be selected to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein to minimize interactions with other members of the receptor complex.
• transmembrane domain of zeta, eta, FceRI gamma, or CD3-gamma, CD3-delta or CD3-epsilon to retain physical association with other members of the receptor complex.
• the extracellular domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction.
• the extracellular domain may be part of a protein which is monomeric, homodimeric, heterodimeric, or associated with a larger number of proteins in a non-covalent complex.
• the extracellular domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions, or may become covalently associated with other Ig heavy/light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains.
• the heavy/light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct.
• the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed.
• Single-chain antibodies are desirable for ease of manipulation.
• the most widely known scAb is one where the variable regions of the heavy and light chain are tethered by a molecular linker so that the tripartite molecule folds spontaneously to form the relevant antigen-binding domain.
• Other forms of single-chain antibodies are contemplated to fall within the scope of the invention.
• scAb's are desirable because a gene thereof can be subcloned in the proper operative relationship with the signal sequence, transmembrane domain and cytoplasmic domains to yield a chimeric molecule of interest.
• Antibodies to TAG-72 are preferred, with monoclonal antibodies being more preferred.
• a scAb directed to TAG-72 is a desired antibody derivative in the practice of the instant invention.
• Hrg human Heregulin
• EGF Epidermal Growth Factor
• Her 2 which is expressed on the surface of breast carcinoma cells and ovarian carcinoma calls.
• the murine equivalent is the "Neu" protein (Bargman et al., Nature 319:226-230 (1986)).
• the extracellular domain of Hrg could be joined to the zeta transmembrane and cytoplasmic domains to form a chimeric construct of the invention to direct T cells to kill breast carcinoma cells.
• hybrid extracellular domains can be used.
• the extracellular domain may consist of a CD receptor, such as CD4, joined to a portion of an immunoglobulin molecule, for example the heavy chain of Ig. See W096/24671.
• a receptor is a molecular complex of proteins, where only one chain has the major role of binding to the ligand, it will usually be desirable to use solely the extracellular portion of the ligand binding protein.
• the extracellular portion may complex with other extracellular portions of other proteins or form covalent bonding through disulfide linkages, one may also provide for the formation of such dimeric extracellular region.
• truncated portions thereof may be employed, where such truncated portion is functional.
• the extracellular region of CD4 one may use only those sequences required for binding of gpl20, the HIV envelope glycoprotein.
• Ig is used as the extracellular region
• a few amino acids at the joining region of the natural protein may be deleted, usually not more than 10, more usually not more than 5.
• the deletion or insertion of amino acids will usually be as a result of the needs of the construction, providing for convenient restriction sites, ease of manipulation, improvement in levels of expression, or the like.
• the cytoplasmic domain as already indicated will generally be from about 10 to 500 amino acids, depending upon the particular domain employed.
• the transmembrane domain will generally have from about 25 to 50 amino acids, while the extracellular domain will generally have from about 10 to 500 amino acids.
• the ⁇ ignal sequence at the 5' terminus of the open reading frame (ORF) which directs the chimeric protein to the surface membrane will be the signal sequence of the extracellular domain.
• ORF open reading frame
• the signal sequence will be removed from the protein, being processed while being directed to the surface membrane, the particular signal sequence will normally not be critical to the subject invention.
• associated with the signal sequence will be a naturally occurring cleavage site, which will also normally be the naturally occurring cleavage site associated with the signal sequence or the extracellular domain.
• the instant invention is particularly directed to single-chain antibody (scAb) chimeric receptors in which a scAb functions as the extracellular domain of the chimeric receptor although other antibody portions can be found at the extracellular domain of a chimera of interest.
• scAb single-chain antibody
• the scAb chimeric receptors function by bypassing the normal antigen recognition component of the T cell receptor complex, and transducing the signal generated on antigen-receptor binding directly via the cytoplasmic domain of the molecule.
• a range of scAb chimeric receptors for example, anti-TAG-72 immunoglobulin-zeta (Ig- ⁇ ) chimeric receptors can be configured.
• the full-length IgG heavy chain comprising the VH, CHI, hinge, CH2 and CH3 (Fc) Ig domains is fused to the cytoplasmic domain of the zeta chain via the appropriate transmembrane domain. If the VH domain alone is sufficient to confer antigen-specificity (so-called "single-domain antibodies”) , homodimer formation of the Ig- ⁇ chimera is expected to be functionally bivalent with regard to antigen binding sites.
• both the IgH- molecule and the full-length IgL chain are introduced into cells to generate an active antigen-binding site. Dimer formation resulting from the intermolecular Fc/hinge disulfide bonds results in the assembly of Ig-C receptors with extracellular domains resembling those of IgG antibodies. Derivatives of this Ig ⁇ C chimeric receptor include those in which only portions of the heavy chain are employed in the fusion. For example, the VH domain (and the CHI domain) of the heavy chain can be retained in the extracellular domain of the Ig-C chimera (VH- ⁇ ). Co-introduction of a similar chimera in which the V and C domains of the corresponding light chain replace those of the Ig heavy chain (VL- ⁇ ) can then reconstitute a functional antigen binding site.
• Single-chain antibody variable fragments in which the C-terminus of one variable domain (VH or VL) is tethered to the N-terminus of the other (VL or VH, respectively, (see Figure 1) via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al. (1990) J. Biol. Chem. 265:18615; Chaudhary et al . (1990) Proc. Natl. Acad. Sci. 87:9491).
• the Fv's lack the constant regions (Fc)- present in the heavy and light chains of the native antibody.
• the extracellular domain of the single-chain Ig chimeras consists of the Fv fragment which may be fused to all or a portion of the constant domains of the heavy chain, and the resulting extracellular domain is joined to the cytoplasmic domain of, for example, zeta, via an appropriate transmembrane domain that will permit expression in the host cell, e.g. , zeta, CD4.
• the resulting chimeric molecules differ from the Fv's in that on binding of TAG-72 the receptors initiate signal transduction via the cytoplasmic domain. In contrast, free antibodies and Fv's are not cell-associated and do not transduce a signal on TAG-72 binding.
• the ligand binding domain of the scAb chimeric receptor may be of two types depending on the relative order of the VH and VL domains: VH-l-VL or VL-l-VH (where "1" represents the linker) (See Figure 1).
• the chimeric construct which encodes the chimeric protein according to the instant invention will be prepared in conventional ways. Since, for the most part, natural sequences may be employed, the natural genes may be isolated and manipulated, as appropriate, so as to allow for the proper joining of the various domains. Thus, one may prepare the truncated portion of the sequence by employing the polymerase chain reaction (PCR), using appropriate primers which result in deletion of the undesired portions of the gene. Alternatively, one may use primer repair, where the sequence of interest may be cloned in an appropriate host. In either case, primers may be employed which result in termini, which allow for annealing of the sequences to result in the desired open reading frame encoding the chimeric protein. Thus, the sequences may be selected to provide for restriction sites which are blunt-ended, or have complementary overlaps. During ligation, it is desirable that hybridization and ligation does not recreate either of the original restriction sites.
• PCR polymerase chain reaction
• the extracellular domain may also include the transcriptional initiation region, which will allow for expression in the target host.
• the transcriptional initiation region which may allow for constitutive or inducible expression, depending upon the target host, the purpose for the introduction of the subject chimeric protein into such host, the level of expression desired, the nature of the target host, and the like.
• promoters have been described in the literature, which are constitutive or inducible, where induction may be associated with a specific cell type or a specific level of maturation. Alternatively, a number of viral promoters are known which may also find use. Promoters of interest include the ⁇ -actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus promoter, and the Friend spleen focus-forming virus promoter. The promoters may or may not be associated with enhancers, where the enhancers may be naturally associated with the particular promoter or associated with a different promoter.
• the sequence of the open reading frame may be obtained from genomic DNA, cDNA, or be synthesized, or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, one may wish to use cDNA or a combination thereof. In many instances, it is found that introns stabilize the mRNA. Also, one may provide for non-coding regions which stabilize the mRNA.
• a termination region will be provided 3' to the cytoplasmic domain, where the termination region may be naturally associated with the cytoplasmic domain or may be derived from a different source.
• the termination regions are not critical and a wide variety of termination regions may be employed without adversely affecting expression.
• the various manipulations may be carried out in vitro or may be introduced into vectors for cloning in an appropriate host, e.g., E. coli.
• an appropriate host e.g., E. coli.
• the resulting construct from joining of the DNA sequences may be cloned, the vector isolated, and the sequence screened to insure that the sequence encodes the desired chimeric protein.
• the sequence may be screened by restriction analysis, sequencing, or the like.
• the sequence Prior to cloning, the sequence may be amplified using PCR and appropriate primers, so as to provide for an ample supply of the desired open reading frame, while reducing the amount of contaminating DNA fragments which may have substantial homology to the portions of the entire open reading frame.
• the target cell may be transformed with the chimeric construct in any convenient manner. Techniques include calcium phosphate-precipitated DNA transformation, electroporation, protoplast fusion, biolistics, using DNA-coated particles, transfection, and infection, where the chimeric construct is introduced into an appropriate virus, particularly a non-replicative form of the virus, or the like.
• vectors Once the target host has been transformed, usually, integration, will result. However, by appropriate choice of vectors, one may provide for episomal maintenance.
• a large number of vectors are known which are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell.
• Illustrative vectors include SV40, EBV and BPV.
• constructs will be designed so as to avoid their interaction with other surface membrane proteins native to the target host. Thus, for the most part, one will avoid the chimeric protein binding to other proteins present in the surface membrane. In order to achieve this, one may select for a transmembrane domain which is known not to bind to other transmembrane domains, one may modify specific amino acids, e.g. substitute for a cysteine, or the like.
• the transformed host is capable of expressing the chimeric protein as a surface membrane protein in accordance with the desired regulation and at a desired level, one may then determine whether the transmembrane protein is functional in the host to provide for the desired signal induction. Since the effect of signal induction of the particular cytoplasmic domain will be known, one may use established methodology for determining induction to verify the functional capability of the chimeric protein.
• TCR binding results in the induction of CD69 expression.
• a chimeric protein having a zeta cytoplasmic domain where the host cell is known to express CD69 upon activation, one could contact the transformed cell with the prescribed ligand and then assay for expression of CD69.
• ancillary signals are not required from other proteins in conjunction with the particular cytoplasmic domain, so that the failure to provide transduction of the signal may be attributed solely to the inoperability of the chimeric protein in the particular target host.
• target hosts may be employed, normally cells from vertebrates, more particularly, mammals, desirably domestic animals or primates, particularly humans.
• the subject chimeric constructs may be used for the investigation of particular pathways controlled by signal transduction, for initiating cellular responses employing different ligands, for example, for inducing activation of a particular subset of lymphocytes, where the lymphocytes may be activated by particular surface markers of cells, such as neoplastic cells, virally infected cells, or other diseased cells, which provide for specific surface membrane proteins which may be distinguished from the surface membrane proteins on normal cells.
• the cells may be further modified so that expression cassettes may be introduced, where activation of the transformed cell will result in secretion of a particular product.
• specific agents such as interferons, TNF's, perforans, naturally occurring cytotoxic agents, or the like, where the level of secretion can be greatly enhanced over the natural occurring secretion.
• the cells may be specifically directed to the site using injection, catheters, or the like, so as to provide for localization of the response.
• the subject invention may find application with cytotoxic lymphocytes (CTL), Natural killer cells (NK), TIL's or other cells which are capable of killing target cells when activated.
• CTL cytotoxic lymphocytes
• NK Natural killer cells
• TIL's or other cells which are capable of killing target cells when activated.
• diseased cells such as cells infected with HIV, HTLV-I or II, cytomegalovirus, hepatitis B or C virus, mycobacterium avium, etc., or neoplastic cells, where the diseased cells have a surface marker associated with the diseased state may be made specific targets of the cytotoxic cells.
• the cells may serve as therapeutic agents.
• a receptor extracellular domain e.g., CD4 which binds to a surface marker of the pathogen or neoplastic condition, e.g., gpl20 for HIV
• the cells may serve as therapeutic agents.
• the cells will be able to avoid recognition by the host immune system as foreign and can therefore be therapeutically employed in any individual regardless of genetic background.
• one may isolate and transfect host cells with the subject constructs and then return the transfected host cells to the host.
• suitable host cells include hematopoietic stem cells, which develop into cytotoxic effector cells with both myeloid and lymphoid phenotype including granulocytes, mast cells, basophils, macrophages, natural killer (NK) cells and T and B lymphocytes.
• hematopoietic stem cells which develop into cytotoxic effector cells with both myeloid and lymphoid phenotype including granulocytes, mast cells, basophils, macrophages, natural killer (NK) cells and T and B lymphocytes.
• NK natural killer
• the zeta subunit of the T cell receptor is associated not only with T cells, but is present in other cytotoxic cells derived from hematopoietic stem cells. Three subunits, zeta, eta and the gamma chain of the Fee receptor, associate to form homodimers as well as heterodimers in different cell types derived from stem cells.
• zeta and eta form both homodimers and heterodimers in T cells (Clayton et al. (1991) Proc. Natl. Acad. Sci. USA 88:5202) and are activated by engagement of the cell receptor complex; zeta and the gamma chain of the Fee receptor form homodimers and heterodimers in NK cells and function to activate cytotoxic pathways initiated by engagement of F c receptors (Lanier et al. (1991) J. Immunol. 146:1571; the gamma chain forms homodimers expressed in monocytes and macrophages (Phillips et al. (1991) Eur. J. Immunol.
• zeta will form heterodimers with gamma, it is able to couple to the intracellular machinery in the monocytic lineage; and zeta and the gamma chain are used by IgE receptors (FcRI) in mast cells and basophils (Letourneur et al. (1991) J. Immunol. 147:2652) for signalling cells to initiate cytotoxic function.
• FcRI IgE receptors
• stem cells transplanted into a subject via a method such as bone marrow transplantation exist for a lifetime
• a continued source of cytotoxic effector cells is produced by introduction of the chimeric receptors of the invention into hematopoietic stem cells to fight virally infected cells, cells expressing tumor antigens, or effector cells responsible for autoimmune disorders.
• introduction of the chimeric receptors into stem cells with subsequent expression by both myeloid and lymphoid cytotoxic cells may have certain advantages in patients with multiple or congenital carcinoma expressing TAG-72.
• the chimeric receptor constructs of the invention can be introduced into hematopoietic stem cells followed by bone marrow transplantation to permit expression of the chimeric receptors in all lineages derived from the hematopoietic system.
• High titer retroviral producer lines are used to transduce the chimeric receptor constructs, for example ⁇ -TAG-72/C, into both murine and human T cells and human hematopoietic stem cells through the process of retroviral-mediated gene transfer as described by Lusky et al. in (1992) Blood 80:396.
• the bone marrow is harvested using standard medical procedures and then processed by enriching for hematopoietic stem cells expressing the CD34 antigen as described by Andrews et al. in (1989) J. Exp. Med. 169:1721. The cells then are incubated with the retroviral supernatants in the presence of hematopoietic growth factors such as stem cell factor and IL-6.
• hematopoietic growth factors such as stem cell factor and IL-6.
• the bone marrow transplant can be autologous or allogeneic, and depending on the disease to be treated, different types of conditioning regimens are used (see, Surgical Clinics of North America (1986) 66:589).
• the recipient of the genetically modified stem cells can be treated with total body irradiation, chemotherapy using cyclophosphamide, or both to prevent the rejection of the transplanted bone marrow.
• chemotherapy using cyclophosphamide, or both to prevent the rejection of the transplanted bone marrow.
• no pretransplant therapy may be required because there is no malignant cell population to eradicate and the patients cannot reject the infused marrow.
• gancyclovir may be administered to reduce the percentage of cells expressing the chimeric receptors.
• the multi-drug resistance gene can be included (Sorrentino et al. (1992) Science 257:99) which functions as a preferential survival gene for the marked cells in the patients if the patient is administered a dose of a chemotherapeutic agent such as taxol. Therefore, the percentage of marked cells in the patients can be titrated to obtain the maximum therapeutic benefit from the expression of the universal receptor molecules by different cytotoxic cells of the patient's immune system.
• PCR The polymerase chain reaction, PCR (Mullis et al. (1986) "Cold Spring Harbor Symposium on Quantitative Biology", NY, 263-273) was used to amplify the extracellular and transmembrane portion of CD8 ⁇ (residues 1-187) from pSV7d-CD8 ⁇ and the cytoplasmic portion of the human chain (residues 31-142 from pGEM3C» Some DNA's were obtained from (Littman et al. (1985) Cell 40:237-246; CD8) and (Weissman et al. (1988) Proc. Natl. Acad. Sci. 85:9709-9713; ⁇ ) .
• Plasmids pSV7d-CD8 ⁇ and pGEM3zC were kindly provided by Drs. Dan Littman and Julie Turner (Univ. of CA, S.F.) and Drs. R.D. Klausner and A.M. Weissman (N.I.H.), respectively.
• Primers encoding the 3' sequences of the CD8 fragment and the 5' sequences of the zeta fragment ( ⁇ ) were designed to overlap such that annealing of the two products yielded a hybrid template. From this template the chimera was amplified using external primers containing Xbal and BamHI cloning sites.
• the CD8/C chimera was subcloned into pTfneo (Ohashi et al. (1985) Nature 316:606-609) and sequenced via the Sanger dideoxynucleotide technique (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-
• Antibodies C305 and Leu4 mAb's recognize the Jurkat T, ⁇ chain and an extracellular determinant of CD3 e, respectively.
• OKT8 acquired from the ATCC, recognizes an extracellular epitope of CD8.
• the anti-C rabbit antiserum, #387 raised against a peptide comprising amino acids 132-144 of the murine ⁇ sequence (Orloff et al. (1989) J. Biol. Chem. 264:14812-14817), was kindly provided by Drs. R.D. Klausner, A.M. Weissman and L.E. Samelson.
• the anti-phosphotyrosine mAb, 4G10 was a generous gift of Drs. D. Morrison, B.
• Druker, and T. Roberts. W6/32 recognizes an invariant determinant expressed on human HLA Class 1 antigens.
• the human leukemic T cell line Jurkat and its derivative J.RT3-T3.5 were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) glutamine, penicillin and streptomycin (Irvin Scientific). Chimera-transfected clones were passaged in the above medium with the addition of Geneticin (GIBCO, Grand Island, NY) at 2 mg/ml. Electroporation of pTfneo-CD8/ into Jurkat and J.RT3-T3.5 was performed in a Bio-Rad Gene Pulser using a voltage of 250V and a capacitance of 960 ⁇ F with 20 ⁇ g of plasmid per 10 7 cells.
• NP40 Nonidet P40
• NP40 Nonidet P40
• 150 mM NaCl 150 mM
• 10 mM Tris pH 7.8 in the presence of protease inhibitors, 1 mM PMSF, aprotinin, and leupeptin.
• Lysis buffer for lysates to be analyzed for phosphotyrosine content was supplemented with phosphatase inhibitors as described (Desai et al. (1990) Nature 348:66-69).
• Iodinated lysates were supplemented with 10 mM iodoacetamide to prevent post-lysis disulfide bond formation.
• Digitonin lysis was performed in 1% Digitonin, 150 mM NaCl, 10 mM Tris pH 7.8, 0.12% Triton X-100. After 30 min. at 4°C, lysates were centrifuged for 10 min. at 14,000 rpm. , then precleared with fixed Staphylococcus aureus (Staph A; Calbiochem-Behring) . Alternatively, lysates of cells stimulated with antibody prior to lysis were precleared with sepharose beads. The precleared lysates were -incubated with Protein A Sepharose CL-4B beads which had been prearmed with the immunoprecipitating antibody. Washed immunoprecipitates were resuspended in SDS sample buffer +/- 5% ⁇ -mercaptoethanol and boiled prior to electrophoresis on 11% polyacrylamide gels.
• the medium was aspirated, and the cells lysed in 100 ⁇ l of NP40 lysis buffer. Lysates were precleared, then ultracentrifuged and samples resolved by SDS PAGE.
• cells were coated with the indicated antibodies at saturating concentrations (1:1000 dil. of ascites) for 30 min. at 4°C. After removal of unbound antibody, cells were spun onto 24-well tissue culture plates which had been precoated with rabbit anti-mouse Ig (Zymed Labs) and blocked with medium plus 10% FBS. Phorbol myristate acetate, PMA (Sigma) and ionomycin (Calbiochem) were added to final concentrations of 10 mg/ml and 1 mM, respectively. Cell-free supernatants were harvested after 20 hr.
• the CD8/C chimeric construct described previously was transfected via electroporation into both the Jurkat human T cell leukemic line, yielding clone JCD8/ ⁇ 2, and a Jurkat-derived mutant, JRT3.T3.5 deficient in full length T s ⁇ chain transcripts and protein, yielding J ⁇ -CD8/ ⁇ l4.
• JRT3.T3.5 expresses normal levels of T j a and the CD3 subunits, its deficiency in T j ⁇ expression results in the absence of TCR expression on the cell surface (Ohashi et al. (1985) Nature 316:606-609).
• Transfection of the chimera into this cell enabled assessment of ⁇ ' s signalling phenotype without the complication of the additional TCR chains.
• Levels of surface expression of the chimera and TCR in stably transfected clones were quantified by flow cytometry using mAb's which recognize either CD8 (OKT8) or the CD3 e subunit of the TCR (Leu 4). Fluorescence histograms of these clones which both express high levels of CD8/C was observed; this cell was used as a control in all of the experiments.
• the three clones express comparable levels of CD8 epitopes and T cell receptors with the exception of J ⁇ -CD8/zl4, which fails to express surface TCR.
• the CD8/ ⁇ chimera can be expressed on the cell surface in the absence of the TCR chains.
• CD8/C chimeric protein To characterize the structure of the CD8/C chimeric protein, cells were surface radioiodinated, lysed in 1% NP40 and subjected to immunoprecipitation with OKT8 or normal rabbit antiserum raised to a cytoplasmic peptide sequence of murine ⁇ .
• any protein forming a heterodimer with CD8/C is likely to form one with the wild-type CD8 and thus should not account for any signalling events specifically attributable to the CD8/ ⁇ chimera.
• Non-covalent association of the chimera with endogenous CD3 gamma ( ⁇ ), delta ( ⁇ ), and epsilon (e) may complicate the interpretation of signals transduced by the chimera.
• ⁇ endogenous CD3 gamma
• delta delta
• e epsilon
• TCR-expressing chimera-transfectant JCD8/ ⁇ 2 show identical patterns characteristic of a CD3 (Leu 4) immunoprecipitate.
• TCR-associated ⁇ is not well iodinated, as its extracellular domain contains no tyrosine residues for labelling, ⁇ immunoblots of CD3 immunoprecipitates confirm its presence under such lysis conditions.
• a small quantity of labelled CD3 e is seen in the Leu 4 immunoprecipitate of the TCR deficient cell despite the fact that this same mAb failed to stain this cell.
• the small amount of immunoprecipitated protein seen is likely due to radiolabelling of internal CD3 e in a small number of permeabilized or non-viable cells during the labelling procedure.
• ⁇ immunoblot analysis was performed comparing ⁇ and OKT 8 immunoprecipitates in Jurkat CD8 and JCD8/ ⁇ 2.
• the anti- ⁇ antiserum immunoprecipitates both the chimera and from JCD8/ ⁇ 2, but only endogenous ⁇ from the Jurkat CD8 control.
• OKT8 immunoprecipitates the chimera but not ⁇ in JCD8/C2, while neither species is detected in Jurkat CD8.
• C305 has no effect in the TCR-negative transfectant, J ⁇ -CD8/ ⁇ l4.
• Stimulation of the chimera on both JCD8/C2 and J ⁇ -CD8/ ⁇ l4 with OKT8 results in the appearance of a pattern of tyrosine-phosphorylated bands indistinguishable from that seen with TCR stimulation.
• stimulation through wild-type CD8 in Jurkat does not result in induction of tyrosine phosphoproteins .
• the CD8/C chimera in the absence of T, and CD3 v, ⁇ , and e, is capable of activating the tyrosine kinase pathway in a manner analogous to that of an intact TCR.
• JCD8/2 expresses two discernible forms of ⁇ on its surface, endogenous ⁇ and the CD8/ ⁇ chimera, each of which could be stimulated independently, the specificity of receptor-induced ⁇ phosphorylation was addressed.
• Immunoprecipitates of ⁇ derived from the three clones were analyzed by western blotting with an anti-phosphotyrosine antibody. A small fraction of the ⁇ immunoprecipitates were blotted with ⁇ antiserum to control for differences in protein content between samples. Analysis of the lysate derived from TCR-stimulated Jurkat CD8 cells reveals a typical pattern of ⁇ phosphorylation with the multiplicity of bands from 16-21 kD most likely representing the varying degree of phosphorylation of the seven cytoplasmic tyrosine residues of £ .
• T cell activation results from the delivery of receptor-mediated signals to the nucleus where they act to induce expression of specific genes.
• One such gene encodes the activation antigen CD69, whose surface expression is induced within hours of T cell receptor stimulation and appears to be dependent on activation of protein kinase C (Testi et al., J. Immunol. 142:1854-1860).
• CD69 protein kinase C
• Flow cytometry reveals a very small degree of basal CD69 expression on unstimulated cells. Maximal levels are induced on all cells with phorbol myristate acetate, PMA, an activator of protein kinase. Stimulation of the TCR results in induction of CD69 on Jurkat CD8 and JCD8/C2, but not on the TCR-negative clone, J ⁇ -CD8/Cl4. Moreover, stimulation of cells with OKT8 induces CD69 on both cells expressing the CD8/ chimera. Though a minimal degree of CD69 induction is apparent with stimulation of wild-type CD8 protein, this level is no higher than that observed with stimulation of Jurkat CD8 with a Class I MHC antibody w6/32.
• IL-2 interleukin-2
• the IL-2 gene is tightly regulated, requiring the integration of a number of signals for its transcription, making, it a valuable distal market for assessing signalling through the CD8/C chimera. Stimulation of Jurkat CD8 and JCD8/C2 cells with TCR antibodies in the presence of PMA results in production of IL-2.
• JCD8/C2 and Jurkat CD8 cells were stimulated with the indicated mAb or inomycin (1 ⁇ m) in the presence of PMA (10 ng/ml).
• IL-2 secretion was determined by the ability of culture supernatants of stimulated cells to support the growth of the IL- ⁇ dependent CTLL-2.20 cells. Since PMA alone induces no IL-2 production in Jurkat, yet has a small direct effect on the viability of the CTLL 2.20 cells, values obtained with PMA alone were subtracted from each response value, yielding the numbers shown above Data from two independent experiments are presented.
• JCD8/2 results in levels of secreted IL-2 consistently higher than those produced in that cell with TCR stimulation.
• J ⁇ -CD8/Cl4 responded more weakly to all experimental stimuli in this assay, but the data were qualitatively similar in that this cell reproducibly secreted IL-2 in response to OKT8 but not to C305.
• CD4-Zeta Chimeric Receptor In Signal Transduction Construction of CD4-zeta Chimeras
• Plasmid pGEM3zeta bears the human zeta cDNA and was provided by Dr. R.D. Klausner and Dr. S.J. Frank (NIH, Bethesda, MD).
• the plasmid pBS.L3T4 bears the human CD4 cDNA, and was provided by Dr. D. Littman and Dr. N. Landau (University of California San Francisco, CA) .
• a BamHI-Apal restriction fragment (approximately 0.64 kb) encompassing the entire human zeta chain coding sequence from residue 7 of the extracellular (EXT) domain, was excised from pGEM3zeta, and subcloned into the BamHI and Apal restriction sites of the polylinker of pBluescript II SK (+) 9pSK is a phagemid based cloning vector from Stratagene (San Diego, CA), generating pSK.zeta.
• Single-stranded DNA was prepared from pSK.CD4.zeta (Stratagene pBluescript II protocol), and used as a template for oligonucleotide-mediated directional mutagenesis (Zoller & Smith (1982) Nucleic Acids Res. 10:6487-6500) to generate CD4-zeta chimeras with the desired junctions described below.
• CD4-zeta fusions 1, 2, and 3 were subsequently sequenced via the Sanger dideoxynucleotide technique (Sanger et al. (1977) Proc. Natl. Acad. Sci. 74:5463-5467), excised as EcoRI-Apal restriction fragments and cloned into the polylinker of expression vector pIK.1.1 or pIK.l.l.Neo at identical sites.
• An EcoRI-BamHI restriction fragment (approximately 1.8 kb) encompassing the entire coding region of CD4 was excised from pSK.CD4.zeta, and subcloned between the EcoRI and Bglll sites of the pIK.1.1 or pIK.l.l.Neo polylinker.
• the plasmid pUCRNeoG (Hudziak et al. (1982) Cell 31:137-146) carries the neomycin gene under the transcriptional control of the Rous Sarcoma virus (RSV) 3' LTR.
• the RSV-neo cassette was excised from PURCNeoG as a Hindi restriction fragment (app. 2.3 kb), and subcloned between the two Sspl sites of pIK.1.1, generating pIK.l.l.Neo.
• pIK.1.1 is a mammalian expression vector constructed by four successive cassette insertions into pMF2, which was created by inserting the synthetic polylinker 5 '-Hindlll-Sphl-EcoRI-Aatll- BglI-XhoI-3' into Kpnl and Sad sites of pSKII (Stratagene), with loss of the Kpnl and Sad sites.
• a BamHI-Xbal fragment containing the SV40 T antigen polyadenylation site (nucleotides 2770-2533 of SV40, Reddy et al.
• Nhel-Sall fragment containing the SV40 origin of replication (nucleotides 5725-5578 of SV40) were inserted by three-part ligation between the Bglll and Xhol sites, with the loss of the Bglll, BamHI, Xbal, Nhel, Sail and Xhol sites.
• the BamHI-Xbal and Nhel-Sall fragments were synthesized by PCR with pSV2Neo (Southern & Berg (1982) J. Mol. Appl. Gen. 1:327-341) as the template using appropriate oligonucleotide primer pairs which incorporated BamHI, Xbal, Nhel and Sail sites at their respective ends.
• an Sphl-EcoRI fragment containing the splice acceptor of the human al globin gene second exon was inserted between the Sphl and EcoRI sites.
• the Sphl-EcoRI fragment was synthesized by PCR with p ⁇ SV ⁇ HP (Treisman et al. (1983) Proc. Natl. Acad. Sci. 80:7428-7432) as the template using appropriate oligonucleotide primer pairs, which incorporated Sphl and EcoRI sites at the respective ends.
• the synthetic polylinker 5 '-EcoRI-Bglll-ApaI-AatII-3' was inserted between the EcoRI and the Aatll sites.
• Hindlll-Sacl fragment containing the CMV IE enhancer/prompter (nucleotides -674 to -19, Boshart et al. (1985) Cell 41:521-530) and a Sacl-Sphl fragment containing the CMV IE first exon/splice donor (nucleotides -19 to +170) were inserted by three-part ligation between the Hindlll and Sphl sites.
• the Hindlll-Sacl fragment was prepared by PCR with pUCH.CMV (M. Calos, Stanford University, Palo Alto, CA) as the template using appropriate oligonucleotide primers which incorporated Hindlll and Sad sites at the respective ends.
• the Sacl-Sphl fragment was chemically synthesized.
• CD4-zeta chimeric receptors were constructed from the extracellular (EC) and cytoplasmic (CYT) domains of CD4 and zeta respectively.
• the transmembrane (TM) domains of the CD4-zeta receptors were derived from zeta (FI, F2) or CD4 (F3).
• F2 and F3 possess all four V domains.
• FI retains only the VI and V2 of the CD4 EC domain (residues 1-180 of the mature CD4 protein), the TM domain of zeta (residues 8-30 of the mature zeta chain) and the CYT domain of zeta (residues 31-142 of the mature zeta chain).
• F2 retains the CD4 EC domain comprising all four V regions (residues 1-370 of the mature CD4 protein), the TM domain of the zeta chain (residues 8-30 of the mature zeta chain) and the CYT domain of zeta (residues 31-142 of the mature zeta chain).
• F3 retains the CD4 EC domain comprising all four V domains (residues 1-371 of the mature CD4 protein), the TM domain of CD4 (residues 372-395 of the mature CD4 chain), and the CYT domain of zeta (residues 31-142 of the mature zeta chain) .
• Chimeric receptors FI, F2, and F3, and the native CD4 gene were introduced into an expression vector pIK.1.1 which directs transcription via the CMV promoter/enhancer.
• pIK.1.1 directs transcription via the CMV promoter/enhancer.
• Constructs were introduced by electroporation into the human embryonic kidney cell line, 293
• OKT4A Although similarly high levels of surface F2 and F3 were detected by OKT4A, the level of FI detected by the antibody in the same transient assay was extremely low.
• FI was present in the membrane, and to assess the structure of the chimeric proteins, immunoprecipitation of radiolabelled proteins was carried out. Twenty hours after electroporation of 293 cells with either FI, F2 or F3, cells were pulse-labelled with 35 S-methionine for four hours, lysed in 1% NP40, and subjected to immunoprecipitation by either 0KT4A (Ortho Pharmaceuticals, NJ) or a rabbit antiserum raised against a cytoplasmic peptide of murine zeta (obtained from R. Klausner, NIH, MD).
• 0KT4A Ortho Pharmaceuticals, NJ
• a rabbit antiserum raised against a cytoplasmic peptide of murine zeta obtained from R. Klausner, NIH, MD.
• Native zeta exists as a disulfide-linked homodimer or as a heterodimer in which the zeta chain is associated with an alternatively spliced product of the same gene, Eta.
• FI and F2 both possess the TM domain of zeta, and therefore should have the potential to form a homodimer (and possibly a heterodimer with native zeta) via the membrane proximal cysteine residue (position 11 of the mature zeta chain).
• the transmembrane domain of F3 is derived from CD4, and would therefore be expected to confer the native monomeric state of the native CD4 molecule to the F3 receptor.
• FI and F2 gave rise to proteins of approximately 70 kd and 150 kd, respectively under non-reducing conditions, approximately double that seen under reducing conditions (approximately 34 kd and 70 kd respectively).
• the results demonstrate that FI and F2, like native zeta, exist as disulfide-linked homodimers, whereas F3 exists as a monomer, as does native CD4. The data do not rule out the ability of F3 to form a noncovalently associated dimer.
• CD4-zeta Receptors into a Human T Cell Line
• the chimeric receptor genes FI, F2, and F3, and the native CD4 gene were introduced into a derivative of pIK.1.1 bearing a selective marker, pIK.l.lNeo.
• Each construct was stably introduced via electroporation into the human T cell leukemia line, Jurkat, and independent Jurkat clones obtained by limiting dilution and selection of G418.
• Cell surface expression of the chimeric receptor was assessed by FACS analysis of Jurkat clones employing FITC-coupled OKT4A.
• FACS analysis of over 100 Jurkat clones revealed that the F3 receptor has the potential to be stably expressed in Jurkat cells at significantly higher levels (up to 50-fold) than the F2 receptor.
• CD69 (Leu-23) is an early human activation antigen present on T, B, and NK lymphocytes. CD69 is detected on the cell surface of T lymphocytes within 2 hours after stimulation of CD3/TCR, reaching a maximal level by 18 to 24 hours. CD69 is therefore the first detectable cell surface protein induced in response to CD3/TCR-mediated signals, and represents a reliable marker of T cell activation.
• the ability of the CD4-zeta chimeric receptors to specifically mediate CD69 induction in the Jurkat T cell line was investigated. Representative Jurkat clones expressing either F2, F3, or CD4 were selected for functional analysis.
• Monoclonal antibodies specific for the Ti a/ ⁇ or CD3 chains can mimic the effect of antigen and serve as agonists to stimulate signal transduction and T cell activation events.
• Cells were stimulated with immobilized mAb's specific for (a) the T, ⁇ chain Jurkat, (C305), (b) the CD3 e chain (OKT3), and (c) the VI domain of CD4 (OKT4A) .
• W6/32 recognizes an invariant determinant of human HLA class 1 antigens, and was used in some experiments as negative control.
• CD69 expression was assayed by FACS analysis approximately 18 hours post-stimulation, employing FITC-couples anti-Leu 23 mAb. Unstimulated cells exhibited a very low level of basal CD69 expression but upon stimulation with a pharmacological activator of protein kinase C, phorbol myristate acetate (PMA), maximal expression was induced. Stimulation of native Ti with the C305 mAb, or native CD3 with the OKT3 mAb, also resulted in induction to the CD69 marker. However, stimulation by OKT4A gave rise to a high level of CD69 expression only for those transfectants expressing a chimeric CD4-C receptor. Indeed, for a number of transfectants, particularly F3-derived, the level of CD69 induction observed upon stimulation was equal to that seen with PMA.
• PMA protein kinase C
• CD4 chimeric receptors possessing the cytoplasmic tail of zeta function effectively in initiation of T cell activation events.
• chimeric CD4-zeta receptors bearing the CD4 TM domain (F3) mediate T cell activation more efficiently (with respect to CD69 induction) than those bearing the zeta TM domain (F2), despite the fact that the latter retains the homodimeric form of native zeta.
• F3 differs from F2 and native zeta, in that it does not exist in the form of a covalent homodimer. The data therefore demonstrate that covalent dimerisation of the chimeric receptor is not essential for initiation of T cell activation as measured by CD69 induction.
• the chimeric receptor cc49-zeta is composed of several subunits: an scFv consisting of the humanized VH and VL regions from the cc49 murine antibody that binds the TAG-72 antigen, linked to the gamma 1 hinge and CH3 domains of human IgG, the human CD4 transmembrane domain, and the human CD3-zeta intracellular domain ( Figure 2).
• the VH and VL regions are joined by an synthetic (Gly 4 -Ser) 3 linker that has been used in several scFv antibodies and is retained from the original humanized cc49 antibody.
• pRT43.2 is a derivative of the pIK vectors discussed hereinabove and disclosed in W094/29438.
• the vectors carry MMLV gag sequences to improve packaging and the Xhol-Clal fragment of pZIPneoSVX is deleted.
• the EcoRI-Apal polylinker from pIKl.l was inserted downstream of the splice acceptor to enable transfer of inserts from pIK plasmids into retroviral constructs.
• the resulting plasmid is called pRTD1.2 and contains both 5' and 3' MMLV LTR's.
• the 5' LTR U3 region of pZIPneoSVX (Cepko et al. (1985) Cell 37:1053-1062) was replaced with the MMSV U3, derived from the Hindlll/Sacl fragment of pIKMMSV, to generate pRTD4.2.
• pRTD2.2SVG was constructed by replacing the 750 bp
• pRTD2.2SSA was constructed by replacement of the 1441 bp SacI-EcoRI fragment of pRTD2.2 with the 1053 bp SacI-EcoRI fragment of LXSN.
• pRTD2.2SVGE- was constructed by synthesis of an oligo encoding bases 2878-2955 of pLXSN (GenBank Accession #M28248) which had been appended by addition of an Apal site at the 5' end. That was used to replace the Apal-Nhel fragment of pRTD2.2SVG, which contains the DNA sequence 3' of the polylinker and 5' of the Nhel site in the 3' LTR.
• pIKT2.2SVG was constructed by insertion of a SacI-EcoRI fragment, which contains part of the HCMV promoter at the 5' end and includes an additional 750 bp downstream from the 3' LTR, between the Sad and EcoRI sites of pIKl.l.
• pIKT2.2SVGE-F3 was constructed by replacing the 182 bp Apal-Nhel fragment of pIKT2.2SVGF3 with the 80 bp Apal-Nhel fragment from pRTD2.2SVGE-F3.
• pRT43.2F3 was derived from pIKT2.2SVGE-F3 by replacing the EcoRI-Apal polylinker downstream from the 3 ' LTR with a synthetic oligo containing an Ascl site. Also, the Ndel site at the 3 ' end of the viral gag sequences was converted to an Xhol site by oligo insertion.
• pRT43.3PGKF3 was derived from pRT43.2F3 first by removal of the 3 ' LTR and insertion of a 3 ' LTR in which the sequences from PvuII to Xbal were deleted (MMLV, GenBank accession #J02255, nucleotide numbers 7938-8115). In addition the MMLV splice acceptor region was replaced with a human phosphoglycerate kinase gene promoter (GenBank Accession #M11958, nucleotides 2-516) which was cloned into a polylinker with an Xhol site at the 5' end and EcoRI site at the 3' end.
• Retroviral Vector with PGK Enhancer Driven cc49-z ⁇ ta with Ig ⁇ CH2 (OlYu ? Mutated to Ala) (pRT43.3PGKHuCC49Fvg237aINTl)
• a PGK enhancer driven cc49-g237a-zeta retroviral vector (pRT43.3PGKHuCC49Fvg237aINTl) containing the V H and V L regions of the humanized single-chain cc49 linked to the human Ig ⁇ l and Ig ⁇ 2 CH2 (Gly ⁇ , mutated to Ala), Ig ⁇ 2 CH3 and CD3 zeta domains was obtained.
• a 1212 bp Ncol-Smal fragment containing the humanized single-chain cc49 scFv was excised from the pTAHuCC49SCLgdCHl vector, a PCR clone of the humanized cc49 scAb (provided by J.
• pRT43.3PGKCC49dCH2 A PGK enhancer driven cc49-g237a-zeta retroviral vector (pRT43.3PGKCC49dCH2) containing the V H and V L regions of the humanized single-chain cc49 linked to the human Ig ⁇ l hinge and Ig ⁇ lCH3 and zeta domains was created.
• pRT43.3PGKHuCC49Fvg237aINTl was digested with Rsrll and Nsil to yield an 8243 bp vector fragment.
• the construct was completed by an oligonucleotide linker that had Rsrll and Nspl ends.
• Retroviral Vector with PGK Enhancer Driven cc49-zeta with Ig ⁇ l CH3 Domain pRT43.2LTRCC49dCH2
• An MMLV LTR enhancer driven cc49-g237a-zeta retroviral vector pRT43.2LTRCC49dCH2 , containing the V H and V L regions of the humanized single-chain cc49 linked to the human Ig ⁇ l hinge and Ig ⁇ lCH3 and zeta domains was made.
• pRT43.3PGKCC49dCH2 was digested with EcoRI and Apal to yield an 1834 bp EcoRI-Apal fragment containing the cc49 scFv which was ligated to a 6702 bp EcoRI-Apal fragment containing the retroviral vector and MMLV LTR enhancer sequences from pRT43.2F3, an MMLV LTR enhancer driven CD4 (Vl,V2,V3,V4)-zeta retroviral vector.
• Retroviral Vector with PGK Enhancer Driven cc49-zeta with Ig ⁇ 2 CH2 (Gly ⁇ - Mutated to Ala) (pRT43.3PGKCC49g237a)
• a PGK enhancer driven cc49-g237a-zeta retroviral vector (pRT43.3PGKg237aCH2) , containing the V H and V L regions of the humanized single-chain cc49 linked to the human Ig ⁇ 2 CH2 (Gly ⁇ - mutated to Ala), Ig ⁇ 2 CH3 and zeta was obtained.
• PGKHuCC49Fvg237aINTl was digested with Rsrll and Nsil to yield an 8243 bp vector fragment. Into that was cloned the gamma2 hinge and the g237a mutation as a 622 bp Nsil-Hinpl fragment from pRT43.3PGKF25g237a. The construct was completed by an oligo linker that had Rsrll and Hinpl ends.
• a PGK enhancer driven cc49-g237a-zeta retroviral vector (pRT43.2LTRg237a) , containing the V H and V L regions of the humanized single-chain cc49 linked to the human Ig ⁇ 2 CH2 (Gly ⁇ mutated to Ala), Ig ⁇ 2 , CH3 and zeta was constructed.
• the 6702 bp EcoRI to Apal retroviral vector fragment from pRT43.2F3 was ligated to the cc49 scFv containing 2167 bp EcoRI to Apal fragment from pRT43.3PGKCC49g237a in a two part ligation.
• T cell cc49-zeta expression was stable. Over the course of 35 days of continuous culture (the length of the experiment was that long), the expression of cc49-zeta did not decrease as, determined by FACS.
• TAG-72 Most of the cell lines examined to date are negative for TAG-72 expression. That result is consistent with the observation that few gastrointestinal-derived tumor cell lines are TAG-72 + in vitro and those that are express low heterogeneous levels of TAG-72 (Hand et al. (1985) Cane. Res. 45:833-840).
• the levels of TAG-72 expressed by cultured LS174T cells were several logs lower than that observed when those cells were maintained in vivo in nude mice and are lower than levels of anti-TAG-72 staining observed with patient samples.
• cc49-zeta + CD8 T lymphocytes were extremely potent killers of Jurkat cells (Proc. Natl. Acad. Sci. (1991) 88:2037-2041) with significant target cell specific lysis observed at effector to target (E:T) ratios as low as 0.3:1 ( Figure 3, top left panel).
• cc49-zeta + human T lymphocytes did not kill another human T cell line CCRF-CEM (Cancer Res. (1967) 27:772-783) ( Figure 3, top right panel).
• Non-transduced donor effectors were used as a control for non-specific lysis.
• the specific cytolytic capacity of cc49-zeta CD8 + T lymphocytes was tested with a series of TAG-72 positive and negative gastrointestinal tract-derived tumor cell lines.
• the positive cell lines were NCI H508 (Cancer Res. (1987) 47:6710-6718) (cecum adenocarcinoma) ( Figure 4, top left panel), LS-174T (colon adenocarcinoma) ( Figure 4, top center panel) and LS-180 (Cancer Res. (1967) 45:833-840) (colon adenocarcinoma) ( Figure 4, top right panel).
• cc49-zeta + CD8 + T lymphocytes (closed circles) lysed all TAG-72 positive cell lines and the level of cytolysis correlated with H508, LS-174T and LS-180 target cell TAG-72 expression levels (Table A) .
• Non-transduced CD8 + lymphocytes were included as a control for non-specific lysis (closed squares).
• TAG-72 + LS-174T target cells Lysis of TAG-72 + LS-174T target cells was not observed with either CD8 T lymphocytes expressing an irrelevant chimeric scFv-zeta receptor, F25, directed against HIVgpl20env or non-transduced T cells. Furthermore, cc49-zeta CD8 T cells did not lyse syngeneic T cells.
• cc49-zeta CD8 + lymphocytes did not lyse any of the TAG-72' gastrointestinal carcinoma cell lines tested.
• the TAG-72 negative cell lines included MIP (colon carcinoma), SNU-1 (Cane.
• the target specificity of cc49-zeta CD8 + T lymphocytes was tested with several cell lines derived from such tumor types.
• KLE-B cells express heterogeneous levels of TAG-72 by FACS, there was extensive lysis of KLE-B cells by cc49-zeta CD8 + T lymphocytes. No significant lysis of TAG-72 negative breast and lung carcinoma-derived cell lines by cc49-zeta CD8 + T lymphocytes was observed. Non-transduced ND1 donor lymphocytes were included as a control for non-specific lysis.
• Cytolytic activity of cc49-zeta CD8+ lymphocytes Absence of bystander target cell lysis
• TAG-72 expression was down-regulated following in vitro culture of TAG-72 + tumor . cell lines (Hand et al. (1985) Cane. Res. 45:833-840). As Jurkat cells expressed constitutive high levels of TAG-72 (similar to primary tumor samples), Jurkat cells were used in the subsequent studies to assay the different of cc49-zeta human T lymphocytes activities.
• TAG-72 + cancer cells While cc49-zeta CD8 + T lymphocytes mediated only minor cytolysi ⁇ of TAG-72 negative cell lines, in patients, TAG-72 + cancer cells will be adjacent to TAG-72" normal tissue. Therefore an important measure of the specificity of cc49-zeta T cells is the absence of lysis of TAG-72" targets when cultured with TAG-72 + targets.
• TAG-72 + and 51 Cr-labeled TAG-72 " cells was set up to address whether lysis of the antigen positive targets resulted in the non-specific lysis of antigen negative bystander cells. No increase in non-specific cc49-zeta T cell mediated lysis was observed following coculture of increasing numbers of unlabelled (cold) TAG-72 + Jurkat cells (closed circles) in the presence of 5l Cr-labeled TAG-72' Snu-1 gastric adenocarcinoma cells (open circles) ( Figure 5).
• Oligonucleotide-mediated deletion mutagenesis was used to generate chimeric receptors with the following compositions:
• CD4 extracellular and transmembrane domain CD4 amino acids 1-395
• CD3 ⁇ cytoplasmic domain CD3 ⁇ amino acids 117-160
• CD4 extracellular domain CD4 amino acids 1-370
• CD3 ⁇ transmembrane and cytoplasmic domains CD3 ⁇ amino acids 83-160
• CD4 extracellular and transmembrane domain CD4 amino acids 1-395
• CD3 ⁇ cytoplasmic domain CD3 ⁇ amino acids 107-150
• CD4 extracellular domain CD4 amino acids 1-370
• CD3 ⁇ transmembrane and cytoplasmic domains CD3 ⁇ amino acids 73-150
• CD4-CD3e (i) CD4 extracellular and transmembrane domain (CD4 amino acids 1-395) and CD3e cytoplasmic domain (CD3e amino acids 132-185);
• CD4 extracellular domain CD4 amino acids 1-370
• CD3e transmembrane and cytoplasmic domains CD3e amino acids 98-185.
• hematopoietic stem cells By engineering, hematopoietic stem cells, a multi-lineage cellular immune response can be mounted against the disease target, such as, cancers expressing TAG-72. After transduction of stem cells followed by bone marrow transplantation, the engineered bone marrow stem cells will continually produce the effector cells abrogating the need for ex vivo cell expansion. Because stem cells are self-renewing, once transplanted, these cells can provide lifetime immunologic surveillance with applications for chronic diseases such as malignancy.
• Effector cells including T cells, neutrophils, natural killer cells, mast cells, basophils and macrophages are derived from hematopoietic stem cells and utilize different molecular mechanisms to recognize the targets.
• T cells recognize targets by binding of the T cell receptor to a peptide in the groove of a MHC molecule on an antigen presenting cell.
• the chimeric receptors of the invention can bypass the MHC-restricted T cell receptor in T cells.
• Other cytotoxic cells of the immune system recognize targets through F c receptors.
• F c receptors bind to the F c portion of antibody molecules which coat virally infected, fungally infected, or parasite infected cells.
• ADCC antibody dependent cellular cytotoxicity
• the transduction method used for introducing the chimeric receptors into stem cells was essentially the same as described in Finer et. al. (1994) Blood 83:43-50.
• 293 cells transfected with the thymidine kinase gene were plated at 10 5 cells/well in a Corning 6-well plate. The cells serve as transient viral producers.
• CD34 + cells were isolated from low density mononuclear human bone marrow cells using a CellPro LC34 affinity column (CellPro, Bothell, WA) .
• Recovered cells were plated out in Myelocult H5100 media (Stem Cell Technologies Inc., Vancouver, B.C.) containing 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL-6 and 10"* M hydrocortisone for a period of 48 hours for "pre-stimulation" .
• the 293/TK cells were transfected as described by Finer et. al., supra.
• the CD34 + cells were collected and resuspended in infection media consisting of IMDM, 10% FBS, Glutamine, 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL-6 and 8 ⁇ g/ml polybrene.
• 3-5 x 10 5 cells were added in 2 ml total to each well of the transfected 293 cells to initiate the co-culture.
• CD34 + cells Forty-eight hours later the CD34 + cells were collected. Briefly, the 2 mis of cell supernatant were removed and additional adherent CD34 + cells were dislodged using an enzyme free/PBS based cell dissociation buffer. Cells were then expanded and differentiated in vitro in Myelocult medium with addition of 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL-6, and 10 ⁇ M Gancyclovir to inhibit 293 proliferation. The cells will not survive under gancyclovir selection, due to carrying the thymidine kinase gene.
• Cells were monitored via cytospins and differentials to ascertain the degree of differentiation and maturity of the neutrophils. Between days 16-24, the cells can be used for testing effector functions such as cytotoxicity, and ascertaining the degree of transduction by FACS and PCR analysis.
• the differentiated neutrophils express the CD15 antigen, and the neutrophils derived from transduced stem cells also express the human CD4 extracellular domain (derived from CD4-zeta) .
• the human CD4 extracellular domain derived from CD4-zeta
• approximately 18% of the neutrophils were expressing CD4-zeta, and the correction was factored in the calculation of effector:target ratio.
• the cytotoxicity of the neutrophils was tested according to the following methods. Cytotoxicity Assay
• Raji target cells expressing the envelope protein of HIV (gpl60), were labeled with sodium 51 Cr chromate (Amersham, Arlington Heights, IL) , generally 50 ⁇ Ci/10 6 cells for 2 hours. The targets were then washed 3 times to remove loosely bound 51 Cr, and resuspended at 10 5 cells/ml in RPMI1640, 10% FBS, and glutamine.
• Modified CD34-derived neutrophils expressing the CD4-zeta chimeric receptor, were plated in triplicate and titrated 1:2 in a final volume of 100 ⁇ l.
• the E:T ratio is dependent on the cell number available, but usually was in the range of 100-200:1.
• a 100 ⁇ l portion (10,000 cells) of the target cell solution was added to each well. Plates were then spun for 2 minutes at 500 RPM and then allowed to incubate for 5 hours at 37°C and 5% C ⁇ 2 . 51 Cr released in the supernatant was counted using a ⁇ counter.
• a chimeric protein which may serve as a surface membrane protein, where the extracellular domain is associated with a ligand of interest, while the cytoplasmic domain, which is not naturally associated with the extracellular domain, can provide for activation of a desired pathway.
• cells can be transformed so as to be used for specific purposes, where cells will be activated to a particular pathway by an unnatural ligand. That can be exemplified by using CD4 as the extracellular domain, where binding of an HIV protein can result in activation of a T cell which can stimulate cytotoxic activity to destroy infected cells.
• other cells may be modified, so as to be more effective in disease treatment, or to immune effects and the like.
• Human natural killer (NK) cells can be genetically modified to express high levels of CD4 using retroviral transduction.
• the CD4 ⁇ chimeric receptor is biochemically active, as cross-linking of CD4 ⁇ on NK cells results in tyrosine phosphorylation of CD4£ and multiple cellular proteins.
• the CD4 ⁇ chimeric receptor is functionally active, and can direct NK cells to specifically and efficiently lyse either natural killer-resistant tumor cells expressing the relevant ligand, gpl20, or CD4 + T cells infected with HIV.
• NK3.3 clone The human NK3.3 clone has been previously described in Kornbluth et al. (1982) J. Immunol. 129:2831.
• Cells were maintained in NK media: RPMI 1640 supplemented with 15% fetal bovine serum, glutamine, penicillin, streptomycin and 15% Lymphocult-T (Biotest, Denville, NJ) .
• Cell density was maintained at less than 1 x 10 6 cells/ml, and media were replaced every two days.
• Retroviral transduction of NK3.3 cells was carried out employing the kat retroviral producer system previously described for transduction of CD8 + T lymphocytes (Roberts et al. (1994) Blood 84:2878 and Finer et al. (1994) Blood 83:43) with the following modifications. 293 cells were plated at 1 x 10 6 cells per plate in a 6-well plate with 2 ml of media per well (293-1), and 48 hours later were transiently transfected with 10 ug of retroviral vector encoding CD4C, pRTD2.2F3 and 10 ug of packaging plasmid. 24 hrs post transfection, media were replaced with NK media.
• NK3.3 cells were added per transfected 293-1 plate and co-cultivated in the presence of polybrene (2 ug/ml). After a 24 hour cocultivation period, NK3.3 cells were removed from the 293-1 plate, and subjected to a second round of co-cultivation with freshly transfected 293 cells for an additional 24 hrs. Transduced NK3.3 cells were then harvested and allowed to recover for 24 to 48 hrs in NK media. Stable expression of the CO4 ⁇ chimeric receptor in transduced NK3.3 was analyzed 15 days post transduction by flow cytometry with FITC-conjugated anti-CD4 mAb's as described below. CD4C + NK cells were subsequently purified by immunoaffinity anti-CD4 mAb-coated flasks (Applied Immune Sciences).
• Anti-Fc ⁇ RIII mAb 3G8 was from Medarex (West Riverside, NH) ; anti-CD4 mAb OKT4A was from Ortho Diagnostic Systems (Raritan, NJ); sheep affinity purified F(ab') 2 fragments to mouse IgG; biotin-conjugated F(ab') 2 fragment goat anti-mouse IgG were from Cappel (Durham, NC) ; anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology (Lake Placid, NY); anti- ⁇ rabbit anti-serum, #387, raised against a peptide comprising amino acids 132-144 of the human ⁇ sequence, was kindly provided by Dr. L. E.
• FITC conjugated-antibodies, Gammal, anti-CD16 (-Fc ⁇ RIII), and anti-CD4 OKT4A mAb's were obtained from Becton-Dickinson (San Jose, CA) .
• Rabbit anti-human lymphocyte serum was from Accurate Chemical and Scientific Corp. (Westbury, NY).
• Anti-gpl20 mAb was from Dupont/NEN Research Products (Wilmington, DE); allophycocyanin streptavidin was from Molecular Probes, (Eugene, OR).
• MOPC 21 (IgG,), used as a control mAb in three colored FACS analysis, and goat serum were from Sigma (St. Louis, MO) .
• Anti-human class II (HLA-DP) mAb was from Becton Dickinson (San Jose, CA) . Sheep anti-mouse Ig peroxidase, donkey anti-rabbit Ig peroxidase, and the ECL western blotting system were from Amersham (Arlington Heights, IL) .
• NK3.3 and CD4 ⁇ + NK3.3 cells were fasted in RPMI 1640 containing 1 mg/ml BSA for 2-3 hrs prior to stimulation. Cells were then spun down and resuspended in the same medium at a density of 2 x 10 7 cells/ml. The cell suspensions were incubated with mAb to Fc ⁇ RIIIA (3G8) or CD4 (OKT4A) for 15 minutes at 4°C, and then washed to remove unbound antibody. Sheep affinity purified F(ab') 2 fragments to mouse IgG were then added at 37°C for 3 minutes in order to cross-link Fc ⁇ RIIIA or CD4 ⁇ .
• cells were lysed at 2 x 10 7 cells/200 ml of 1% NP-40, 150 mM NaCl, and 10 mM Tris (pH 7.8) in the presence of protease inhibitors (1 mM PMSF, aprotinin, leupeptin) , and phosphatase inhibitors (0.4 mM EDTA, NaHC ⁇ 3 , 10 mM Na 4 P 2 0 7 10H 2 O) . After 30 minutes at 4°C, lysates were centrifuged for 10 minutes at 14,000 rpm, and pre-cleared with protein A Sepharose beads.
• the pre-cleared lysates were then incubated with the immunoprecipitating anti- ⁇ serum at 4°C for 30 minutes, followed by protein A Sepharose beads at 4°C overnight. Washed immunoprecipitates were then subjected to SDS-PAGE under reducing conditions.
• FITC fluorescein isothiocyanate
• HIV-gpl20 expression was analyzed by staining with mouse anti-gpl20 mAb or isotype negative control, followed by incubation with goat anti-mouse biotin F(ab ' ) 2 , followed by allophycocyanin-streptavidin prior to analysis. Allophycocyanin-stained cells were then analyzed using a Becton Dickinson Facstar Plus.
• Cytotoxicity was determined using a standard 4 hr chromium-51 ( 51 Cr) release assay (Matzinger (1991) Immunol. Methods
• CPM cpm released by targets incubated with effector cells
• MR cpm released by targets lysed with 100 ⁇ l of 1% triton x-100 (i.e., maximum release)
• SR cpm released by targets incubated with medium only (i.e. spontaneous) .
• the CEM.NKR human T cell line is described in Byrn et al. (1990) Nature 344:667.
• the JAM test was employed for measuring cell lysis (Matzinger 1991), and is based on the amount of [ 3 H]thymidine labeled DNA retained by living cells. In brief, 1 x 10* actively proliferating target cells were labeled with 20 uCi [ 3 H]thymidine overnight.
• Raji is a human B cell lymphoma which expresses high levels of class II MHC.
• Raji cells expressing low levels of HIV env were generated by co-transfection with the expression vector, pCMVenv, which encodes rev and env (gpl60) from the HXB2 HIV-1 clone and the selection plasmid, pIKl.lneo which confers resistance to G418 (Roberts et al., 1994).
• G418-resistant clones were isolated and analyzed for expression of the env proteins gpl20 and gpl60 by immunoblotting with an anti-gpl20 mAb.
• Raji clones positive by immunoblotting were then subjected FACS analysis to detect surface expression of gpl20.
• the NK cell line 3.3 was originally isolated from human peripheral blood mononuclear cells (PBL). NK3.3 exhibits an NK characteristic cell surface phenotype (CD3", CD16 + ), and mediates strong natural killer activity.
• the CD4 ⁇ chimeric receptor was introduced into NK3.3 cells by retroviral mediated transduction using the kat packaging system (Finer et al., 1994). After transduction, 26% of the transduced NK population expressed CD4 ⁇ as detected by immunofluorescence of surface CD4. A population in which greater than 85% of the cells expressed high levels of chimeric receptor was obtained after immunoaffinity purification of transduced NK cells with anti-CD4 mAb's. It was noted that nmodified and CD4C ⁇ modified NK3.3 cells express comparable levels of Fc ⁇ RIIIA.
• Both CD4C and native ⁇ were immunoprecipitated from the cell populations by treating cell lysates with anti- ⁇ serum, and the immunoprecipitated supernatants were subsequently analyzed on immunoblots with an anti-phosphotyrosine antibody (4G10) .
• Tyrosine phosphorylation of CD4 ⁇ , but not native ⁇ is rapidly induced by crosslinking of the chimeric ⁇ -receptor on NK cells. That result is consistent with previous studies conducted in T lymphocytes which have shown that cross-linking of chimeric C-receptors induces phosphorylation of the chimeric receptor, but not of native ⁇ present in T cell receptor (TCR)/CD3 complexes.
• TCR T cell receptor
• CD3 T cell receptor
• Fc ⁇ RIIIA is thought to mediate cellular activation through a tyrosine-kinase dependent pathway, as indicated by the results of previous studies demonstrating rapid tyrosine phosphorylation of cellular proteins upon crosslinking of Fc ⁇ RIIIA (Laio et al., 1993; Ting et al., 1992; Azzoni et al., 1992; and Salcedo et al., 1993). Rapid tyrosine phosphorylation of cellular proteins with molecular masses of approximately 136, 112, 97, and 32 kDa is induced upon cross-linking of either Fc ⁇ RIIIA or CD4C receptors on CD4C/NK cells. The sizes of the proteins are similar to those previously reported as undergoing phosphorylation upon cross-linking of Fc ⁇ RIIIA (Liao et al., 1993 and Ting et al., 1992) .
• NK cells For NK cells, similar functional associations between p56 lc and Fc ⁇ RIII have been shown to be mediated through direct interaction with ⁇ (Azzoni et al., 1992 and Salcedo et al., 1993), this subunit also acting as a substrate for p56 ,ck in vitro.
• CD4 ⁇ chimeric receptor is able to activate the tyrosine kinase signaling pathway in a manner analogous to the Fc ⁇ RIIIA/C complex in NK cells, presumably due to retention of functional interactions between such protein kinases and the ⁇ moiety of the chimeric receptor.
• CD4( + NK Cells) Mediate Cytolysis against Natural Killer-resistant Tumor Cells
• CD4 The ability of CD4 to confer NK cells with the ability to kill a NK-resistant tumor cell line expressing low levels of gpl20 was evaluated to assess the anti-tumor potential of NK cells expressing chimeric ⁇ -receptors.
• Target cell lines expressing gpl20 were generated from the NK-resistant human Burkitt lymphoma cell line Raji by co-electroporation of pIKneo and pCMVenv. G418-resistant clones were subsequently isolated and analyzed for stable expression of the HIV env proteins gpl20 and gpl60 by western immunoblotting. To detect surface expression of gpl20, it was necessary to employ a highly sensitive allophycocyanin-streptavidin staining procedure with anti-gpl20 mAb.
• Unmodified and CD4 ⁇ -modified NK cells were functionally evaluated in a cytotoxicity assay against either normal Raji cells or Raji-gpl20 cells as targets, over a range of effector: target ratios.
• CD4£ + NK cells were also tested for their ability to lyse normal Raji cells in the presence of rabbit anti-human lymphocyte serum.
• NK cells expressing CD4C exhibit maximal specific lysis as high as 50% over background levels at effector: target ratios of between 25:1 to 50:1.
• the specific lysis observed is highly sensitive, with values of approximately 20% above background observed at effector:target ratios as low as 0.4:1.
• the efficiency of CD4C-mediated cytolysis appears to be more efficient than Fc ⁇ RIIIA-mediated ADCC, at all effector to target ratios tested.
• CD4C and scAb ⁇ chimeric receptors efficiently redirect primary human CD8 + T lymphocytes to target HIV-infected cells (Roberts et al., 1994). It was therefore of interest to compare the cytolytic activity of CD4C + NK cells to that of human PBMC-derived CD8 + T cells expressing CD4C (CD4C + CD8 + T cells) against the same Raji-gpl20 target cell line.
• the highly efficient cytolytic activity observed for CD4C + NK cells is comparable to that observed for CD4C + CD8 + T cells.
• CD4( + NK Cells) Mediate Cytolysis against HIV-infected T Cells
• CD4 ⁇ + NK cells can mount an efficient cytolytic response against HIV-infected CD4 + T cells.
• the NK-resistant CD4 + T cell line CEM.NKR was infected by HIV-1 IIIB as previously described (Byrn et al. (1990) Nature 344:667).
• CEM uninfected
• CEM/IIIB HIV infected CEM-NKR cells
• specific lysis of the virally infected population was observed at effector:target ratios as low as 1.5:1, with maximal lysis as high as 70% above background occurring at effector:target ratios of 50:1.
• CD4 binds to non-polymorphic sites on MHC Class II molecules
• C ⁇ a chimeric receptor for re-directing NK-mediated cytotoxicity toward HIV-infected cells
• CD4 ⁇ a chimeric receptor for re-directing NK-mediated cytotoxicity toward HIV-infected cells
• Chimeric C-receptors in which the CD4 ligand binding domain is fused to the cytoplasmic domain of the signal transducing subunit ⁇ of Fc ⁇ RIIIA and of TCR, are expressed at high levels on the surface of NK cells on retroviral-mediated transduction.
• the CD4 ⁇ chimeric receptor can direct NK cells to initiate a highly effective cytolytic response against natural killer-resistant tumor cells expressing low levels of the relevant target ligand gpl20, and against natural killer-resistant T cells infected with HIV.
• the cytolytic response is highly sensitive, and appears comparable to that previously observed for CD4 ⁇ + and scAb ⁇ + CD8 + T lymphocytes.
• a humanized cc49 antibody (Shu et al. (1993) Proc. Natl. Acad. Sci. 90:7995-7999 and Kashmiri et al. (1995) Hybridoma 14:461-473) was used to prepare a chimeric receptor.
• a scAb consisting of the V H and V L regions of the mAb joined through a (G 4 -S) 3 linker was used.
• a 788 bp fragment obtained by Ncol and Rsrll digestion of plasmid pTAHUCC49SCldgCHl provided by Kashmiri was ligated to the gl hinge and CH3 domain of human IgG, (residues 221-230 and 341-444) using an oligo linker flanked by Rsrll and Nspl sites.
• the extracellular portion (scAb) was attached to the CD3 intracellular region (residues 31-142 of ⁇ ) through the human membrane associated IgG, Ml transmembrane spanning region and human CD4 transmembrane spanning region (residues 372-395 of CD4) to yield the construct encoding the chimeric receptor.
• the cc49- ⁇ construct was cloned in the retroviral vector rkat43.2 (Finer et al., 1994).
• Virus was generated by transient CaP0 4 transfection of 293 cells (Roberts et al., 1994), with the modification of using two helper plasmids, encoding gag/pol and env, respectively, to reduce the likelihood of generating competent virus through recombination.
• the LS174T, KLE-B, CCRF-CEM and MIP-1 cell lines were obtained from J. Schlom of the NIH.
• the LS180, Snu-1, Jurkat, NCI H716 and H508 cell lines were obtained from the ATCC.
• T cells obtained from PBMC of buffy coats were diluted 1:4 with Ca/Mg-free PBS.
• the cells obtained from a ficoll separation were suspended in a T cell medium (TCM; such as, 1:1 AIM-V and RPMI with HEPES, sodium pyruvate, glutamine, pen-strep and 10% human serum) .
• Monocytes were removed by stationary culture in a flask.
• the non-adherent cells were exposed to anti-CD3 and anti-CD28 antibody-coated Dynal beads in TCM. Following culture for about 48 hours the beads were removed. The stimulated cells then were exposed to IL-2 for about 24 hours. The cells were resuspended in IL-2-containing medium and then mixed with retrovirus containing the cc49- ⁇ construct and polybrene (100 IU/ml and 2 ⁇ g/ml final concentrations). After 24 hours, half of the medium was removed and replaced with fresh virus-containing medium. That step was again repeated 24 hours later. On day 6, the cells were removed from the transduction mix and resuspended in TCM containing IL-2.
• Cytokine expression was determined as described herein and using known methods. Along with IL-2, expression of IL-4, TNF-o, IFN- ⁇ and GM-CSF was determined using commercially available reagents (for example, R & D Systems).
• Soluble TAG-72 inhibition assays were conducted using a 2x concentration of sTAG-72 obtained from bovine submaxillary mucin (Sigma). Cells were exposed to the carbohydrate and then tested for cytotoxic activity as described herein.
• SCID-NOD mice were injected with 1-3 x 10 8 cc49-C expressing or normal T cells either iv with 10 6 KLE-B cells or sc with 10 6 LS174T or KLE-B cells. Mice were monitored daily for development of sc tumors or sacrificed at various time points and examined for internal tumors.
• TAG-72 + cell lines, LS174T, LS180, NCI H508, Jurkat and KLE-B were killed by the transduced cells, whereas TAS-72 " cells, MIP-1, NCI H716, CCRF-CEM and Snu-1 cells, were not lysed.
• KLE-B and LS174T are positive for FAS and FAS L and yet are efficiently killed by the construct-expressing cells.
• the transduced cells also killed primary TAG-72 + tumor cells obtained from patients with advanced colon carcinoma. Labelled TAG-72' cells were not lysed by the transduced cells.
• transduced cells When the transduced cells were cultured in vitro. Stimulated cells expressed substantial levels of GM-CSF, IFN- ⁇ and TNF- ⁇ . In vivo, the transduced T cells were immunoprotective.
• LS174T a human colon cancer cell line, introduced subcutaneously into SCID-NOD mices produce a local palpable tumor in 2-4 weeks. The tumors are large and encapsulated. Injection with transduced cells however, prevented the development of tumors over a period of at least 18 weeks.
• the human endometrial carcinoma-derived KLE-B cell line when injected intraperitoneally, results in the development of multiple tumors in the peritoneal cavity associated with the intestine, liver, spleen, kidney and the site of injection.
• CD4 + cells transduced with a cc49 construct lysed suitable targets CD4 + T cells transduced with the cc49- ⁇ chimeric receptor construct lysed not only Jurkat cells but also LS174T cells and KLE-B cells, but did not lyse the
• TAG-72' cells H716 and H508.
• the transduced cells lyse targets in the same fashion as found in normal cytotoxic T cells, that is, via perforin.
• various inhibitors of the T cell lytic pathway were tested, it was noted that inhibitors of the perforin-mediated pathway of lysis inhibited killing of antigen positive targets.
• anti-FAS L and anti-TNF ⁇ antibodies had a minimal impact on the killing ability of transduced cells.
• concanamycin A resulted in almost a complete blockage of killing.
• the T cells proliferate.
• the cells are co-stimulated and exposed to anti-idiotype antibody or a TAG-72 + target, the T cells do not proliferate or produce substantial amounts of cytokines.
• the co-stimulation mechanism might impose another mechanism on the transduction of signalling.

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