Methods of preparing cell therapy products for the treatment of conditions caused by human papillomavirus 6 or 11 infection

Abstract

Disclosed are methods of selectively expanding a number of T cells each having antigenic specificity for a human papillomavirus (HPV) 6 or 11 antigen expressed by a tissue of a mammal. Also disclosed are methods of preparing a T cell product from an HPV 6 or 11 antigen positive papilloma from a mammal. Methods of treating or preventing a condition caused by a chronic HPV 6 or 11 infection in a mammal are also disclosed.

Description

METHODS OF PREPARING CELL THERAPY PRODUCTS FOR THE TREATMENT OF CONDITIONS CAUSED BY HUMAN PAPILLOMAVIRUS 6 OR 11 INFECTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 709,683, filed October 21, 2024, which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project numbers ZIA BC012067-01 and ZIA BC 012131 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Recurrent Respiratory Papillomatosis (RRP) and anogenital condyloma are conditions caused by chronic infection with human papillomavirus (HPV) 6 or 11. These conditions result in papillomatosis growths that affect the upper aerodigestive tract (in the case of RRP) and the anogenital region (in the case of condylomas). In RRP, papilloma growth in the aerodigestive track can lead to dysphonia, dyspnea and, in some cases, airway obstruction, leading to recurrent pneumonia or pulmonary failure. Current RRP treatment options include repeat surgical debulking or laser ablation procedures to palliate symptoms. However, latent viral infection may persist, resulting in continuous regrowth of papilloma. Repeated procedures to debulk the papilloma can expose patients to anesthetic and surgical risk, and emotional distress.

[0004] Despite advances in treatments for chronic HPV 6 or 11 -driven conditions, such as surgery and / or laser ablation, there are currently no systemic treatment options for these conditions. Accordingly, there exists an unmet need for additional treatments for these conditions.BRIEF SUMMARY OF THE INVENTION

[0005] An aspect of the invention provides a method of preparing a T cell product from a papilloma from a mammal, the method comprising culturing one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days, thereby producing a quantity of viable total nucleated cells (TNC) of at least 25 million, wherein the papilloma is infected with HPV 6 or HPV 11 ; and expanding the number of T cells in the viable TNC using one or both of (i) irradiated allogeneic feeder cells and (ii) irradiated autologous feeder cells and one or both of (iii) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex and (iv) one or more cytokines.

[0006] Another aspect of the invention provides a method of selectively expanding a number of T cells from a papilloma of a mammal, wherein the T cells each have antigenic specificity for a HPV 6 or HPV 11 antigen, the method comprising culturing one or more fragments of the papilloma of the mammal in the presence of one or more cytokines, wherein the papilloma is infected with HPV 6 or HPV 11 ; obtaining T cells from the cultured one or more fragments; inducing antigen presenting cells (APCs) of the mammal to present one or more HPV 6 antigens or one or more HPV 11 antigens; and stimulating, for 10 to 19 days and in the presence of one or more cytokines ex vivo, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 6 antigens or the one or more HPV 11 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 6 or HPV 11 antigen.

[0007] An aspect of the invention provides a method of isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen, the method comprising preparing a T cell product from a papilloma from a mammal according to any of the inventive methods described herein: and isolating a TCR, or an antigen-binding portion thereof, from the expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for an HPV 6 or HPV 11 antigen.

[0008] Another aspect of the invention provides a method of isolating a TCR, or an antigen-binding portion thereof, having antigenic specificity an HPV 6 or HPV 11 antigen, the method comprising selectively expanding a number of T cells from a papilloma of a mammal according to any of the inventive methods described herein; and isolating a TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, whereinthe TCR. or antigen-binding portion thereof, has antigenic specificity for an HPV 6 or HPV 1 1 antigen.

[0009] Still another aspect of the invention provides a method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen, the method comprising isolating a TCR, or an antigen-binding portion thereof, according to any of the inventive methods described herein, and introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into peripheral blood mononuclear cells (PBMC) to obtain cells that express the TCR, or the antigen-binding portion thereof.

[0010] Another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising selectively expanding a number of T cells from a papilloma of a mammal according to any of the inventive methods described herein; and administering to the mammal the selectively expanded number of T cells in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

[0011] Still another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising preparing a T cell product according to any of the inventive methods described herein; and administering to the mammal the T cell product in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

[0012] Another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen according to any of the inventive methods described herein; and administering to the mammal the population of cells that express the TCR, or antigen-binding portion thereof, in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] Figure 1 is a schematic illustrating a method of preparing a T cell product from a papilloma from a mammal in accordance with an aspect of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0014] Tumor infdtrating lymphocyte (TIL) therapy involves the administration of therapeutic T cells isolated and expanded from resected cancer deposits. However, prior to the research described herein, it was not known whether this type of therapy can be obtained from non-malignant tissue, for at least the following reasons. Non-malignant tissue, such as papilloma, are likely to be more genetically stable and have fewer tumor neoantigens as compared to tumor. Moreover, it has been shown that papilloma containing higher expression of HPV 6 / 11 viral antigens (E2, E4, E5gamma, E6 and E7) have higher levels of CXCL8 and neutrophilic myeloid-derived suppressor cells (MDSCs) with low levels of T cell infiltration (Norberg et al.. Set. Transl. Med., 15, eadj0740 (2023)). These neutrophils have been shown to suppress activation of T cells in multiple in vitro assays (Bai et al..Laryngoscope, 134: 3238-3244 (2024)). These data demonstrate that HPV actively suppresses T cell infiltration and activation. Therefore, prior to the research described herein, it was not known whether T cells infiltrating chronically infected, non-malignanttissue such as papilloma could be generated in large enough quantities to be used as a therapeutic for such conditions such as RRP.

[0015] It has now been discovered that papilloma infiltrating lymphocytes (PIL), e.g., T cells, can be obtained to a quantity sufficient for clinical use. A quantity of T cells sufficient for clinical use may be, for example, 30 to 100 billion T cells (e.g., 30 billion, 35 billion. 40 billion, 45 billion, 50 billion, 55 billion, 60 billion, 65 billion, 70 billion, 75 billion, 80 billion, 85 billion, 90 billion, 95 billion, 100 billion, or a range defined by any two of the foregoing values). Surprisingly, the T cells obtained by the inventive methods were able to eliminate papilloma tissue by the end of the manufacturing process, which is not commonly seen in cell manufacturing processes of TIL. These results show that T cells obtained by the inventive methods may be prepared and used for the treatment or prevention of conditions caused by chronic HPV 6 and HPV 11 infections, including RRP, laryngotracheal disease, and anogenital condyloma.

[0016] An aspect of the invention provides a method of preparing a T cell product from a papilloma from a mammal. A papilloma is a benign (non-cancerous) tumor. Tn an aspect of the invention, the papilloma is not a pre-malignancy.

[0017] The method may comprise obtaining a papilloma sample from a mammal. The papilloma sample may be obtained from the mammal in any suitable manner such as, for example, biopsy or surgical resection. In an aspect of the invention, the mammal with the papilloma has a condition caused by a chronic HPV 6 infection or a condition caused by a chronic HPV 11 infection. In an aspect of the invention, the condition is RRP, laryngotracheal disease, or anogenital condyloma. In an aspect of the invention, the condition is not cancerous and is not a pre-malignancy. There are rare instances where RRP can turn into an invasive squamous cell carcinoma of the lung. In an aspect of the invention, the condition is cancerous.

[0018] The papilloma from the mammal may be infected with HPV 6 or HPV 11.Accordingly, the papilloma may comprise HPV 6-infected cells or HPV 11 -infected cells. The HPV 6 or HPV 11 virus infecting the papilloma expresses HPV 6 antigens or HPV 11 antigens.

[0019] The method may comprise testing the papilloma sample for the presence or expression of one or more HPV 6 antigens or one or more HPV 11 antigens. The testing may comprise testing for the expression of any protein (e.g., an antigen) specifically expressed by HPV 6 or HPV 11, expression of any RNA encoding the HPV 6- or HPV- 11 -specific protein, or a combination thereof. Testing for HPV 6 or HPV 11 antigen expression may be carried out in any suitable manner known in the art. Exemplar} tests may include any one or more of reverse transcriptase (RT) polymerase chain reaction (PCR)-based genotyping and Western blots. Examples of HPV 6 antigens include HPV 6 El, HPV 6 E2, HPV 6 E4, HPV 6 E5, HPV 6 E6, HPV 6 E7, HPV 6 LI, and HPV 6 L2. Examples of HPV 11 antigens include HPV 11 El, HPV 11 E2, HPV 11 E4, HPV 11 E5, HPV 11 E6, HPV 11 E7, HPV 11 LI, and HPV 11 L2.

[0020] An aspect of the invention comprises dividing the papilloma sample into multiple fragments. The papilloma sample may be divided into any suitable number of fragments such as, for example, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more fragments (or a range defined by any two of the foregoing values). The papilloma fragment may be divided in anysuitable manner e.g.. mechanically (disaggregating the papilloma using, e.g.. a GENTLEMACS Dissociator, Miltenyi Biotec, Auburn, CA) or enzymatically (e.g., collagenase or DNase).

[0021] An aspect of the invention comprises culturing one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days. The duration of the culture may be, for example, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or a range of defined by any two of the foregoing values, for example, 15 to 28 days, 14 to 27 days, 15 to 26 days, 16 to 25 days, 17 to 24 days, 18 to 23 days, 19 to 22 days, or 20 to 21 days.

[0022] The one or more fragments may be cultured in any container suitable for tissue culture, for example, a multi-well plate, flask, or vessel (e.g., G-REX 6M multi-well cell culture plate or G-REX 10 vessel (Wilson Wolf Manufacturing, LLC, St. Paul, MN)).

[0023] The one or more fragments may be cultured in any medium suitable for papilloma tissue culture. An example of a medium suitable for culturing the papilloma fragments is AIM-V + 10% HI AB serum, 2 mM GLUTAMAX supplement (Thermo Fisher Scientific Inc., Waltham, MA). The culture medium may further comprise one or more antibiotics and / or one or more antifungals (e.g., penicillin, streptomycin and gentamicin).

[0024] The one or more cytokines may comprise, e.g., one or more of interleukin (IL)-2, IL-7. IL-15, and IL-21.

[0025] In an aspect of the invention, the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces a minimum quantity of viable TNC required for the non-selective expansion using the rapid expansion protocol (REP) (described in more detail below). In an aspect of the invention, this minimum quantity of viable TNC required for the non-selective expansion is at least 25 million viable TNC. For example, the quantity of viable TNC produced may be at least 25 million, at least 26 million, at least 27 million, at least 28 million, at least 29 million, at least 30 million, at least 31 million, at least 32 million, at least 33 million, at least 34 million, at least 35 million, at least 36 million, at least 37 million, at least 38 million, at least 39 million, at least 40 million, at least 41 million, at least 42 million, at least 43 million, at least 44 million, at least 45 million, at least 46 million, at least 47 million, at least 48 million, at least 49 million, at least 50 million, at least 51 million, at least 52 million, at least 53 million, atleast 54 million, at least 55 million, at least 56 million, at least 57 million, at least 58 million, at least 59 million, at least 60 million, at least 61 million, at least 62 million, at least 63 million, at least 64 million, at least 65 million, at least 66 million, at least 67 million, at least 68 million, at least 69 million, at least 70 million, at least 71 million, at least 72 million, at least 73 million, at least 74 million, at least 75 million, at least 76 million, at least 77 million, at least 78 million, at least 79 million, at least 80 million, at least 81 million, at least 82 million, at least 83 million, at least 84 million, at least 85 million, at least 86 million, at least 87 million, at least 88 million, at least 89 million, at least 90 million, at least 91 million, at least 92 million, at least 93 million, at least 94 million, at least 95 million, at least 96 million, at least 97 million, at least 98 million, at least 99 million, or at least 100 million, or more.

[0026] In an aspect of the invention, the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces a quantity of T cells of 13 million to 150 million. For example, the quantity of T cells produced may be 13 million. 20 million. 30 million. 40 million. 50 million. 60 million. 70 million, 80 million, 90 million, 100 million, 110 million, 120 million, 130 million, 140 million, 150 million, or a range defined by any two of the foregoing values.

[0027] In an aspect of the invention, the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces at least 80% viable cells. For example, the percentage of viable cells produced may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. For example, the percentage of viable cells produced may be 80% to 99%, 81% to 98%, 82% to 97%, 83% to 96%, 84% to 95%, 85% to 94%, 86% to 93%, 87% to 92%, or 88% to 91 %.

[0028] In an aspect of the invention, the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces at least 55% CD3+ cells. For example, the percentage of CD3+ cells produced may be at least 55%. at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%,at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. For example, the percentage of CD3+ cells produced may be 55% to 99%, 56% to 98%, 57% to 97%, 58% to 96%, 59% to 95%, 60% to 94%. 61% to 93%. 62% to 92%. 63% to 91%. 64% to 90%. 65% to 89%. 66% to 88%. 67% to 87%, 68% to 86%, 69% to 85%, 70% to 84%, 71% to 83%, 72% to 82%, 73% to 81%, 74% to 80%, 75% to 79%, or 76% to 78%.

[0029] In an aspect of the invention, the one or more fragments of the papilloma are regressed or eliminated after the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days. This result was surprising because methods of producing TIL cell products do not typically result in the regression or elimination of the tumor fragments used to prepare the TIL cell products. The regression or elimination of the one or more fragments of the papilloma observed by carrying out the inventive methods supports the use of the T cell products produced by the inventive methods for the treatment or prevention of conditions caused by chronic HPV 6 infection or chronic HPV 11 infection.

[0030] Though preventative HPV vaccination may be highly effective at generating a strong B cell response to prevent infection with low-risk HPV, it has not been consistently shown to be capable of eliminating epithelial cells already infected with HPV, which requires a strong HPV-specific T cell response. Therefore, the inventive methods may be useful for generating a T cell response of sufficient magnitude to regress or eliminate papilloma and latently HPV -infected epithelial cells.

[0031] The method may further comprise expanding the number of T cells (e.g., in the viable TNC) that were produced by culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days. In this regard, the method may further comprise culturing the viable TNC or the T cells in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex (also referred to herein as “non-selective expansion’" or “rapid expansion protocol (REP)"’). Non-selective expansion of the numbers of T cells can be accomplished by any of a number of methods as described in, for example,U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent 11.401,503; Dudley et al.. J.Immunother ., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). The one or more cytokines used in the non-selective expansion may include, for example, one or more of interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin- 15 (IL-15), with IL-2 being preferred. An example of antibody which specifically binds to the human CD3 complex is OKT3 antibody (available from Ortho-McNeiL Raritan, N.J.). The method may comprise carrying out no more than a single round of non-selective expansion or multiple rounds of non-selective expansion. Non-selective expansion may increase the number of T-cells by at least 2-fold (or 3-. 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least 100-fold over a period of a week). The fold expansion achieved may be highly variable and may be donor-dependent.

[0032] The inventive method may produce a quantity of T cells sufficient for clinical use following non-selective expansion. The quantity of T cells sufficient for clinical use that is produced by the inventive method, and following non-selective expansion, may vary from patient to patient. For example, the quantity7of T cells sufficient for clinical use and which is produced following non-selective expansion may be from 30 to 100 billion T cells or more (e.g., 30 billion, 35 billion. 40 billion, 45 billion, 50 billion. 55 billion, 60 billion, 65 billion, 70 billion, 75 billion, 80 billion, 85 billion, 90 billion, 95 billion, 100 billion, or a range defined by any two of the foregoing values). By contrast, the quantity of T cells sufficient for experimental use is a quantity7that would be the minimum sufficient to test the T cells for reactivity, e.g., no more than 2 x 104T cells.

[0033] Another aspect of the invention provides a method of isolating a TCR, or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen. The method may comprise preparing a T cell product from a papilloma from a mammal according to any of the inventive methods described herein.

[0034] The method may further comprise isolating a TCR, or an antigen-binding portion thereof, from the expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity7for an HPV 6 or HPV 11 antigen. The “the antigen-binding portion” of the TCR, as used herein, refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically bindsto the HPV 6 or HPV 11 antigen as described herein with respect to other aspects of the invention. The term '‘antigen-binding portion’’ refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the HPV 6 or HPV 11 antigen, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, 50%, 70%, 80%, 90%, 95%, or more, of the parent TCR.

[0035] The antigen-binding portion can comprise an antigen-binding portion of either or both of the a and chains of the TCR, such as a portion comprising one or more of the complementarity determining region (CDR)l, CDR2, and CDR3 of the variable region(s) of the a chain and / or P chain of the TCR. In an aspect of the invention, the antigen-binding portion can comprise the amino acid sequence of the CDR1 of the a chain (CDRla), the CDR2 of the a chain (CDR2a), the CDR3 of the a chain (CDR3a), the CDR1 of the P chain (CDR1P), the CDR2 of the P chain (CDR2P), the CDR3 of the P chain (CDR3P), or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDRla, CDR2a, and CDR3a; the amino acid sequences of CDRip, CDR2P, and CDR3P; or the amino acid sequences of all of CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of the TCR.

[0036] In an aspect of the invention, the antigen-binding portion can comprise, for instance, the variable region of the TCR comprising a combination of the CDR regions set forth above. In this regard, the antigen-binding portion can comprise the amino acid sequence of the variable region of the a chain (Va). the amino acid sequence of the variable region of the P chain (VP), or the amino acid sequences of both of the Va and VP of the TCR.

[0037] In an aspect of the invention, the antigen-binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen-binding portion can comprise the entire length of the a or p chain, or both of the a and P chains, of the TCR.

[0038] Isolating a nucleic acid comprising a nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the T cells may be carried out in any suitable manner known in the art. For example, the method may comprise isolating RNA from the Tcells and sequencing the TCR. or the antigen-binding portion thereof, using established molecular cloning techniques and reagents such as, for example, 5’ Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR) using TCR-a and -0 chain constant primers.

[0039] The TCR, or the antigen-binding portion thereof, isolated by the inventive methods may be useful for preparing cells for adoptive cell therapies. In this regard, another aspect of the invention provides a method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity' an HPV 6 or HPV 11 antigen. The method may comprise isolating a TCR, or an antigen-binding portion thereof, according to any of the inventive methods described herein.

[0040] The method may further comprise introducing a nucleic acid comprising a nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into PBMC to obtain cells that express the TCR, or the antigen-binding portion thereof.Introducing a nucleic acid comprising the nucleotide sequence (e.g., a recombinant expression vector) encoding the isolated TCR, or the antigen-binding portion thereof, into PBMC may be carried out in any of a variety of different ways know n in the art as described in, e.g., Green et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleic acid comprising a nucleotide sequence into PBMC include transformation, transduction, transfection, and electroporation.

[0041] Another aspect of the invention provides a method of selectively expanding a number of T cells from a papilloma of a mammal, w herein the T cells each have antigenic specificity for a HPV 6 or HPV 11 antigen. It is believed that the inventive method of selective expansion advantageously increases the yield of HPV 6- and HPV 11 -specific T cells. The inventive methods of selective expansion may provide any one or more of a variety of advantages. For example, the inventive methods may provide for the selective expansion of the numbers of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen over the number of T cells which do not have antigenic specificity for a HPV 6 or HPV 11 antigen. The inventive methods may provide populations of cells with a larger proportion of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen as compared to populations of cells prepared by methods which do not selectively expand the number of T cells as described herein. Without being bound to a particular theory ormechanism, it is believed that populations of cells with a larger proportion of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen may provide one or more of improved destruction of target papilloma cells, improved destruction of HPV 6-infected cells, improved destruction of HPV 11-infected cells, treatment of conditions caused by a chronic HPV 6 infection, and treatment of conditions caused by a chronic HPV 11 infection as compared to populations of cells with a smaller proportion of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen. Stimulating T cells having antigenic specificity for a HPV 6 or HPV 11 antigen with APCs that express HPV 6 or HPV 11 antigens may achieve selective growth of HPV 6 or HPV 11 antigen-reactive T cells, which allows generation of T-cell products enriched for HPV 6 or HPV 11 antigen reactivity.

[0042] The method of selective expansion may comprise obtaining a papilloma sample from a mammal, testing the papilloma sample for the presence or expression of one or more HPV 6 antigens or one or more HPV 11 antigens, and dividing the papilloma sample into multiple fragments, all of which may be as described herein with respect to other aspects of the invention.

[0043] The method of selective expansion may comprise culturing one or more fragments of the papilloma of the mammal in the presence of one or more cytokines. The culturing of the one or more fragments of the papilloma of the mammal in the presence of one or more cytokines may be carried out as described herein with respect to other aspects of the invention. In an aspect of the invention, papilloma is infected with HPV 6 or HPV 11, as described herein with respect to other aspects of the invention.

[0044] The method may comprise obtaining T cells from the cultured one or more papilloma fragments. The T cells may be physically isolated from the fragments using routine techniques.

[0045] The method may further comprise inducing APCs of the mammal to present one or more HPV 6 antigens or one or more HPV 11 antigens. The APCs may be autologous to the mammal or synthetic APCs. If synthetic APCs are used, the synthetic APCs may be modified to express one or more HLA restriction elements expressed by the mammal. The APCs may be dendritic cells (DCs); B cells; PBMC; autologous papilloma cells; an HLA-engineered cell line; papilloma organoid cells, or papilloma xenograft cells.

[0046] In an aspect of the invention, inducing the APCs to present the one or more HPV 6 antigens or one or more HPV 11 antigens comprises pulsing the APCs with one or more peptides, wherein the one or more peptides comprise one or more HPV 6 antigen amino acid sequences or one or more HPV 11 antigen amino acid sequences. Examples of HPV 6 antigens and HPV 11 antigens are described herein with respect to other aspects of the invention. In this regard, the APCs may be cultured with one or more peptides in a manner such that the APCs internalize the peptide and display the HPV 6 or HPV 11 antigen amino acid sequence, bound to an MHC molecule, on the cell membrane. Methods of pulsing APCs are known in the art and are described in, e.g.. Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010).

[0047] In an aspect of the invention, inducing the APCs to present the one or more HPV 6 antigens or one or more HPV 11 antigens comprises introducing a nucleotide sequence encoding the one or more HPV 6 antigens or the one or more HPV 11 antigens into the APCs. The nucleotide sequence is introduced into the APCs so that the APCs express and display the HPV 6 or HPV 11 antigen amino acid sequence, bound to an MHC molecule, on the cell membrane. The nucleotide sequence encoding the HPV 6 or HPV 11 antigen amino acid sequence may be RNA or DNA. Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways known in the art as described in, e.g., Solheim et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transformation, transduction, transfection, and electroporation. In an aspect of the invention, the nucleotide sequence introduced into the APCs is PRGN-2012, which is a gorilla adenovirus vaccine expressing a composite protein antigen designed to elicit HPV 6 and HPV 11 T cell responses (Lee et al., NPJ Vaccines. 6: 86 (2021)).

[0048] In an aspect of the invention, inducing the APCs to present the one or more HPV 6 antigens or one or more HPV 11 antigens comprises (i) pulsing the APCs with a peptide comprising an HPV 6 or HPV 11 antigen amino acid sequence or a pool of peptides, each peptide in the pool comprising a different HPV 6 or HPV 11 antigen amino acid sequence; (ii) pulsing the APCs with lysis of the papilloma expressing different HPV 6 or HPV 11 antigens; or (iii) introducing one or more nucleotide sequences encoding one or more different HPV 6 or HPV 11 antigens into the APCs.

[0049] In an aspect of the invention, the one or more HPV 6 antigens or one or more HPV 11 antigens is a plurality of different HPV 6 or HPV 11 antigen amino acid sequences. For example, the number of different HPV 6 or HPV 11 antigen amino acid sequences may comprise amino acid sequences from different HPV antigens (e.g., a (different) amino acid sequence from each one of HPV 6 E6 and HPV 6 E7), different amino acid sequences from the same HPV antigen (e.g., more than one different HPV 6 E7 amino acid sequence), or a combination of any of the foregoing. In an aspect of the invention, the number of different HPV 6 or HPV 11 antigen amino acid sequences in the plurality of different antigen amino acid sequences ranges from 2 to 100 different HPV 6 or HPV 11 antigen amino acid sequences. For example, the number of different HPV 6 or HPV 11 antigen amino acid sequences may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86. 87. 88. 89. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a range defined by any two of the foregoing values. For example, the number of different HPV 6 or HPV 11 antigen amino acid sequences in the plurality of different HPV 6 or HPV 11 antigen amino acid sequences may range from 2 to 100, 20 to 8030 to 70, or 40 to 60, different HPV 6 or HPV 11 antigen amino acid sequences.

[0050] The method may further comprise stimulating, for 10 to 19 days and in the presence of one or more cytokines ex vivo, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 6 antigens or the one or more HPV 11 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 6 or HPV 11 antigen.

[0051] While the stimulating may be carried out without non-selectively expanding the numbers of T cells obtained from the one or more fragments before the stimulation, in an aspect of the invention, the number T cells obtained from the one or more fragments are non-selectively expanded, e.g., as described herein with respect to other aspects of the invention, before the stimulation.

[0052] The stimulating may comprise co-culturing the T cells having antigenic specificity for the HPV 6 or HPV 11 antigen and APCs so that a TCR expressed by the T cells encounters the antigen presented by the APCs in such a manner that the TCR specificallybinds to and immunologically recognizes the antigen presented by the APCs. thereby- initiating proliferation of the T cells having antigenic specificity for the HPV 6 or HPV 11 antigen, and selectively expanding the number of T cells having antigenic specificity for the HPV 6 or HPV 11 antigen over the number of T cells not having antigenic specificity- for the HPV 6 or HPV 11 antigen. In an aspect of the invention, the T cells are co-cultured in direct contact v\dth the APCs.

[0053] The duration of the stimulating of the T cells with the APCs may be from 10 to 19 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or a range defined by any two of the foregoing values, for example, 11 to 18 days, 12 to 17 days, 13 to 16 days, or 14 to 15 days.

[0054] The one or more cytokines used for the stimulating of the T cells with the APCs may be, for example, IL-2, IL-7, IL-15, and IL-21.

[0055] In an aspect of the invention, the method of selectively expanding a number of T cells from the papilloma of the mammal increases the number of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen by 20-fold to 40-fold or more. For example, the inventive methods may increase the number of T cells each having antigenic specificity- for a HPV 6 or HPV 11 antigen by 20-fold, 21-fold, 22 -fold, 23-fold, 24-fold, 25-fold, 26-fold. 27-fold, 28-fold, 29-fold, 30-fold, 31 -fold, 32-fold, 33-fold, 34-fold, 35-fold.36-fold, 37-fold, 38-fold, 39-fold, or 40-fold, or a range defined by any two of the foregoing values, for example, 21- to 39-fold, 22- to 38-fold, 23- to 37-fold, 24- to 36-fold, 25- to 35-fold, 26- to 34-fold, 27- to 33-fold, 28- to 32-fold, or 29- to 31-fold. The foregoing fold expansion may be achieved over a period of 10 to 19 days, for example, 10, 11, 12, 13. 14.15. 16, 17, 18, or 19 days, or a range defined by any two of the foregoing values. The fold expansion achieved by the inventive methods may be highly variable and may be donordependent.

[0056] In an aspect of the invention, the method of selectively expanding a number of T cells from the papilloma of the mammal increases the number of T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen to a number sufficient for clinical use. Numbers of T cells sufficient for clinical use is described herein with respect to other aspects of the invention.

[0057] In an aspect of the invention, the papilloma is infected with HPV 6. the method comprises inducing APCs of the mammal to present one or more HPV 6 antigens; and the method comprises stimulating, for 10 to 19 days and in the presence of one or more cytokines in vitro, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 6 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 6 antigen, as described herein with respect to other aspects of the invention.

[0058] In an aspect of the invention, the papilloma is infected with HPV 11, the method comprises inducing APCs of the mammal to present one or more HPV 11 antigens; and the method comprises stimulating, for 10 to 19 days and in the presence of one or more cytokines in vitro, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 11 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 11 antigen, as described herein with respect to other aspects of the invention.

[0059] In an aspect of the invention, the T cells prepared by (i) the inventive method of preparing a T cell product from a papilloma from a mammal or (ii) the inventive method of selectively expanding a number of T cells from a papilloma of a mammal are oligoclonal. Without being bound to a particular theory or mechanism, it is believed that the oligoclonal nature of the T cells produced by the inventive methods may improve the capacity of the T cells to reduce or eliminate virally infected cells compared to other transferred T cell therapy approaches (e.g., TCR-engineered T cell therapy, which can only recognize one antigen presented by one HLA restriction element). In an aspect of the invention, the T cells prepared by the inventive methods described herein may comprise multiple T cells, each T cell having antigenic specificity for a different HPV 6 antigen or a different HPV 11 antigen. The T cells prepared by the inventive methods described herein may, for example, comprise multiple T cells, each T cell having antigenic specificity for a different HPV 6 antigen amino acid sequence or a different HPV 11 antigen amino acid sequence. Alternatively or additionally, the T cells prepared by the inventive methods described herein comprise multiple T cells, each T cell having antigenic specificity for the HPV 6 antigen or the HPV 11 antigen presented by a different HLA element expressed by the mammal.

[0060] In an aspect of the invention, the T cells prepared by the inventive methods described herein can comprise CD8+T cells, CD4+T cells, or both CD8+T cells and CD4+T cells.

[0061] The inventive methods may, advantageously, provide a population of cells that is enriched for T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen. The population of cells can be a heterogeneous population comprising the T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen together with at least one other cell, e.g., a T cell, which does not have antigenic specificity for a HPV 6 or HPV 11 antigen, or a cell other than a T cell, e g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of (e.g., consisting essentially of) T cells each having antigenic specificity for a HPV 6 or HPV 11 antigen.

[0062] The phrase “antigenic specificity,” as used herein, means that the TCR expressed by the T cell can specifically bind to and immunologically recognize an HPV 6 antigen or an HPV 11 antigen.

[0063] Another aspect of the invention provides a method of isolating a TCR, or an antigen-binding portion thereof, having antigenic specificity an HPV 6 or HPV 11 antigen. The method may comprise selectively expanding a number of T cells from a papilloma of a mammal according to any of the inventive methods described herein. The method may further comprise isolating a TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for an HPV 6 or HPV 11 antigen. Isolating the TCR, or antigen-binding portion thereof, from the selectively expanded number of T cells may be carried out was described herein with respect to other aspects of the invention.

[0064] Another aspect of the invention provides a method of preparing a population of cells that express a TCR. or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen. The method may comprise isolating a TCR, or an antigenbinding portion thereof, from the selectively expanded number of T cells, according to any of the inventive methods described herein. The method may further comprise introducing the nucleotide sequence encoding the isolated TCR. or the antigen-binding portion thereof, intoPBMC to obtain cells that express the TCR. or the antigen-binding portion thereof, as described herein with respect to other aspects of the invention.

[0065] The populations of cells produced by the inventive methods can be isolated and / or purified. The term "isolated," as used herein, means having been removed from its natural environment. The term "purified," as used herein, means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least 50%, can be greater than 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or can be 100%.

[0066] The populations of cells produced by the inventive methods can be formulated into a composition, such as a pharmaceutical composition. In this regard, an aspect of the invention provides a pharmaceutical composition comprising any of the populations of cells described herein and a pharmaceutically acceptable carrier. The inventive pharmaceutical compositions may comprise any of the inventive populations of cells in combination with another pharmaceutically active agent(s) or drug(s).

[0067] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for T cells. Methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in. for example, Remington: The Science and Practice of Pharmacy, 23rdEd., Pharmaceutical Press (2020). It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0068] The choice of carrier may be determined by the particular method used to administer the inventive population of cells. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or interperitoneal administration. More than one route can be used to administer the inventive populations of cells, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0069] Preferably, the population of cells is administered by injection, e.g., intravenously. The pharmaceutically acceptable carrier for cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w / v of NaCl in water, about 300mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water). NORMOSOL R electrolyte solution (Abbott, Chicago, TL), PLASMA-LYTE A injection (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an aspect, the pharmaceutically acceptable carrier is supplemented with human serum albumen. A pharmaceutical composition for infusion may or may not contain IL-2. If the pharmaceutical composition contains IL-2, then a concentration of about 300 lU / mL can be used.

[0070] For purposes of the invention, the amount or dose (e.g., numbers of cells) administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the dose (e.g., numbers of cells) should be sufficient to bind to an HPV 6 or HPV 11 antigen, or treat or prevent a condition in a period of from about 2 hours or longer, e g., 12 to 24 or more hours, from the time of administration. In certain aspects, the time period could be even longer. The dose will be determined by the efficacy of the population of cells and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.

[0071] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or IFN-y is secreted by T cells prepared by the inventive methods upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-y is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0072] The dose of the T cells produced by the inventive methods also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular population of cells. Typically, the attending physician will decide the dosage of the population of cells with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, population of cells to be administered, route of administration, and the severity of the condition being treated. In an aspect, the number of cells administered per infusion may vary', e.g., from 1 x 106to 1 x 1012cells or more. In certain aspects, at least 1.5 x IO10cells may be administered.

[0073] The population of cells produced by the inventive methods may be useful for treating or preventing a condition caused by a chronic HPV 6 or HPV 11 viral infection in a mammal. In an aspect of the invention, the mammal has a chronic HPV 6 infection or a chronic HPV 11 infection. Without being bound to a particular theory' or mechanism, it is believed that T cells from a papilloma prepared according to the inventive methods are likely to treat or prevent a condition caused by a chronic HPV 6 or HPV 11 infection because of any one or more of the following factors: (i) there are no known recurring damaging mutations in, or genomic loss of, genes important for antigen processing and presentation in papilloma like is seen in tumors, so a T cell response may be effective in clearing the infection, (ii) there is consistent expression of HPV viral antigens in papilloma, unlike in tumors, where tumor antigen heterogeneity is commonly seen, and (iii) the condition caused by a chronic HPV 6 or HPV 11 infection is solely driven by the presence of the HPV 6 or HPV 11 virus and its antigens (unlike in cancer, where there tends to be several driver mutations and / or loss of tumor suppressor genes). Additionally, standard of care treatment of these conditions, which is repeat surgical debridement, results in an abundance of papilloma tissue that could be utilized for manufacturing of a cell therapy product.

[0074] Accordingly, another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising: selectively expanding a number of T cells from a papilloma of a mammal according to any of the inventive methods described herein; and administering to the mammal the selectively expanded number of T cells in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

[0075] Another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising preparing a T cell product according to any of the inventive methods described herein; and administering to the mammal the T cell product in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

[0076] Another aspect of the invention provides a method of treating or preventing a condition caused by a chronic viral infection in a mammal, the method comprising preparinga population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen according to any of the inventive methods described herein; and administering to the mammal the population of cells that express the TCR, or antigen-binding portion thereof, in an amount effective to treat or prevent the chronic viral infection in the mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

[0077] In an aspect of the invention, the condition caused by the chronic viral infection is RRP, laryngotracheal disease, or anogenital condyloma. In an aspect of the invention, the RRP is pulmonary RRP. Pulmonary RRP is an understudied clinical entity due to the rarity of the condition and has a high unmet clinical need.

[0078] For purposes of the inventive methods of treating or preventing a condition, wherein cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0079] In an aspect of the invention, the inventive methods of treating or preventing a condition may further comprise administering to the mammal one or more additional treatments for a condition caused by a chronic HPV 6 or infection or chronic HPV 11 infection. Examples of such additional treatments may comprise, but are not limited to, any one or more of a vaccine against HPV 6 or HPV 11. an anti-programmed death-ligand (PD-Ll) antibody, an anti-PD-1 antibody, and an anti-vascular endothelial growth factor inhibitor.

[0080] In an aspect of the invention, the inventive methods of treating or preventing a condition may further comprise administering to the mammal nonmyeloablative lymphodepl eting chemotherapy. The nonmyeloablative lymphodepleting chemotherapy can comprise the administration of one or both of cyclophosphamide (e.g., low-dose cyclophosphamide) and fludarabine. In an aspect of the invention, the nonmyeloablative lymphodepleting chemotherapy is administered prior to administering the cells. In an aspect of the invention, the inventive methods of treating or preventing a condition may further comprise administering to the mammal interleukin (IL)-2.

[0081] As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that themammals are from the order Artiodactyla. including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0082] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a condition caused by a chronic viral infection in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the condition being treated or prevented. For example, the treatment or prevention provided by the inventive method can include promoting the regression of a papilloma. Also, for purposes herein, "prevention" can encompass delaying the onset of the condition, or a symptom or condition thereof.

[0083] The follow ing examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLE 1

[0084] This example demonstrates the identification of T-cells having antigenic specificity for HPV 6 or HPV 11 in T cells infiltrating papilloma.

[0085] It was investigated whether papilloma lack infiltration of HPV-specific T cells. Single-cell RNA sequencing and paired TCR alpha / beta sequencing was performed on papilloma fragments from tw o patients (Patients 1 and 2) not on systemic therapy for RRP. Patient 1 had HPV 6+ disease while Patient 2 had HPV 11+ disease. Using unsupervised clustering analysis, individual cells were clustered based on RNA gene expression. The TCR from cells expressing genes known to be involved in TCR signaling and T cell exhaustion and found previously to associate with tumor-targeting T cells were isolated (Y ossef et al., Cancer Cell 41:2154-2165. e5 (2023)). These TCRs were then cloned into autologous T cells and tested for reactivity against target cells expressing patient-specific, individual HLA alleles along with HPV-type specific, individual viral antigens (E2, E4, E5y, E6 or E7).Reactivity was determined by measuring supernatant concentration of interferon-gamma (IFN-y) and comparing that to IFN-y release from a known TCR that has previously demonstrated clinical activity7, the HPV16 E7 TCR (Nagarsheth et al., Nat. Med., 27:419-425 (2021)). A total of 13 different TCRs targeting four different HPV 6 viral antigens through four different HLA alleles were found in papilloma from Patient 1. A total of 15 different TCRs targeting all five HPV 11 early antigens through four different HLA alleles were discovered in papilloma from Patient 2. These data indicate the presence of HPV 6 or HPV 11 -specific T cells in papilloma capable of targeting multiple different HPV viral antigens through multiple different HLA-restriction elements. These characteristics highlight the potential for papilloma infiltrating lymphocyte (PIL) therapy to target HPV 6 or HPV 11 virally infected cells.EXAMPLE 2

[0086] This example demonstrates a method of preparing a T cell product from a papilloma from a mammal.

[0087] Initial experiments were performed in which T cell products were generated from fragments of resected laryngeal papilloma from four individual patients with RRP who underwent standard of care surgery. In brief, the resected papilloma was collected in Hanks' Balanced Salt Solution (HBSS) with 10 mcg / rnL of gentamicin (Fig. 1). It w as then rinsed in sequential washes of HBSS with 10 mcg / mL of gentamicin to reduce the risk of culture contamination. Tissue dimensions were then recorded, and each papilloma section was cut into fragments of about 1-2 mm3. Various vessels (24-well plates, G-REX 6M multi-well cell culture plate or G-REX 10 vessel (Wilson Wolf Manufacturing, LLC, St. Paul, MN)) were filled with culture media containing AIM-V + 10% HI AB serum, 2 mM GLUTAMAX supplement (Thermo Fisher Scientific Inc., Waltham, MA) and 6,000 lU / mL of IL-2.

[0088] Cultures were then incubated at 37°C and 5% CO2 for 4 - 5 days prior to visual inspection and media exchange. For the media exchange, vessels were observed under an inverted microscope and transferred to the Biological Safety7Cabinet (BSC) in order to avoid disturbing the culture. Half of the media volume was then removed without disturbing the bottom of the culture and replaced with equal volume of fresh media containing AIM-V + 10% HI AB serum, 2 mM GLUTAMAX supplement and 6,000 lU / mL of IL-2. The vesselswere returned to the incubator at 37°C and 5% CO2. Cultures were assessed every 2-3 days to determine the need to feed or split the culture based on visual evaluation. Culture maintenance was performed for a maximum of 28 days. Harvest decision was made based on visible confluency, >90%, by evaluation under an inverted microscope. During harvest, cell expansion and viability- were assessed using an automatic cell counter. Cell phenotype was evaluated using flow cytometry. Remaining cells were cryopreserved for future expansion.

[0089] As shown in Table 1, a median of eight fragments (a range 6 to 50 fragments) were generated from resected papilloma. The number of days required for initial cell culture ranged from 19 to 26 (median 21). which is longer than what is required for the initial phase of TIL manufacturing for melanoma, which is approximately 14 days (Dudley et al., J.Immunother., 26:332-342 (2003)). For manufacturing, a maximum amount of 20 fragments were placed in a single culture vessel, so that the number of fragments used in each single culture vessel ranged from 6 to 20.

[0090] There was no significant difference in the number of viable white blood cells (WBC) / mL or viable TNC based on the number of fragments used in the initial culture. For instance, from patient PD1572-10A, where the number of fragments used was eight, the total viable WBC / mL generated was 4.3e6, which was higher than a culture that contained 20 fragments (PD 1572- 13 (3 A)). In addition, there was no difference in viable WBC / mL and viable TNC based on the initial culture vessel utilized (i.e., 24-well plate vs G-REX 6M multi-well cell culture plate vs. G-REX 10 vessel).

[0091] The median percent viability- from all cultures was 90.2% (range 80.6 to 95.8). Despite the papilloma being infiltrated with fewer T cells as compared to malignant tissue, all patients had initial cell cultures with the necessary quantity of viable cells required for the rapid expansion protocol (REP) phase (i.e., minimum of 25 million viable TNC), where cell numbers needed for therapeutic use can be generated (Dudley et al., J. Immunother., 26:332-342 (2003)).TABLE 1Summas of data for expansion of PIL from resected laryngeal papilloma.Sample PD1572- PD1572-8A PD1572- PD1572- PD1572- PD1572- PD1572- name 8A (HPV (HPV 11+) 9A 10A 13 (1A) 13 (2A) 13 (3A)11+)Days 20 20 26 19 21 21 21 culturedNumber 6 6 6 8 15 15 20 offragmentsInitial 24-well 24-well plate 24-well G-REX G-REX G-REX G-REX culture plate plate 10 vessel 6M plate 6M plate 6M plate vesselViable 1.33E+06 1.20E+06 1.49E+06 4.30E+06 4.17E+05 754E+05 1.02E+06 WBC / mLViable 5.59E+07 2.64E+07 6.41E+07 1.51E+08 1.25E+06 302E+06 2.96E+07 TNCViability 95.5 90.2 95.8 90.4 80.6 84.8 87.3 (%)CD3 (%) 92.19 97.78 92.18 9896 55.59 62.82 91.88 CD4 (%) 99.29 99.76 94.91 9977 24.34 85.45 96.69 CD8 (%) 0.41 012 4.27 0.16 68.13 8.51 1.28 CD56(%) 0.38 062 4.37 0.79 39.2 25.36 2.43

[0092] There were differences in the percentage of CD4+ and CD8+ T cells generated between cultures. For instance, the percentage of CD4+ T cells in cultures from patient PD1572-8A was 99% compared to 24% for culture PD1572-13 1A. Previous experience with TIL therapy for HPV-associated cancer demonstrates a wide range of CD4+ / CD8+ T cell populations, and did not correlate with response to therapy (Stevanovic et al., Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., 25:1486-1493 (2019); Stevanovic et al., J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.. 33:1543-1550 (2015)).

[0093] At least one culture from all four patients had a CD3+ percentage of 90% or greater.

[0094] All cultures grew without the development of visible bacterial or fungal contamination, despite being collected directly from the oropharynx.

[0095] Surprisingly, all papilloma fragments were eliminated by the end of the cell culture, highlighting the ability of the enumerated T cells to eliminate papilloma. This is not typically seen in cultures of TIL, where cancer fragments remain at the end of the initial expansion phase. These data highlight the feasibility of manufacturing PIL infusion products and the ability of PIL to eliminate papilloma in culture.

[0096] To the best of the inventors’ knowledge, this is the first study to demonstrate an ability to generate PILs of sufficient quantity for clinical use. Surprisingly, papilloma fragments regressed by the end of the initial expansion phase, highlighting the potential of this therapy to eliminate papilloma.EXAMPLE 3

[0097] This example demonstrates a method of treating adult patients with RRP with a T cell product from papilloma.

[0098] Adult RRP patients will undergo papilloma debulking surgeries of the upper airway to obtain tissue for generation of autologous PIL as described in Example 2. Patients will receive a conditioning chemotherapy regimen of cyclophosphamide 500 mg / m2 x 3 days and fludarabine 30 mg / m2 x 3 days followed by a single infusion of PIL. Cell infusion will be followed by administration of high-dose aldesleukin (IL-2) for up to 6 doses.

[0099] Clinical and immunologic response will be evaluated 8 weeks following cell infusion. Response and progression from imaging studies (computed tomography (CT) scan or Magnetic Resonance Imaging (MRI)) will be evaluated using the international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) (Eisenhauer et al., Eur. J. Cancer Oxf. Engl. 1990, 45(2):228-247 (2009)). Changes in the largest diameter (unidimensional measurement) of the tumor or cystic lesions will be used in the RECIST criteria.

[0100] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0101] The use of the terms “a” and '‘an’’ and '‘the” and “at least one’’ and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to.”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range,unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary' language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0102] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary- skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly- contradicted by context.

Claims

CLAIM(S):

1. A method of preparing a T cell product from a papilloma from a mammal, the method comprising:culturing one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days, thereby producing a quantity of viable total nucleated cells (TNC) of at least 25 million, wherein the papilloma is infected with human papillomavirus (HPV) 6 or HPV 11; andexpanding the number of T cells in the viable TNC using one or both of (i) irradiated allogeneic feeder cells and (ii) irradiated autologous feeder cells and one or both of (hi) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex and (iv) one or more cytokines.

2. The method of claim 1, wherein the one or more fragments of the papilloma are regressed or eliminated after the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days.

3. The method of claim 1 or 2, wherein the papilloma is infected with HPV 6.

4. The method of claim 1 or 2, wherein the papilloma is infected with HPV 11.

5. The method of any one of claims 1-4, wherein the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces at least 80% viable cells.

6. The method of any one of claims 1-5, wherein the culturing of the one or more fragments of the papilloma ex vivo in the presence of one or more cytokines for 15 to 28 days produces at least 55% CD3+ cells.

7. A method of selectively expanding a number of T cells from a papilloma of a mammal, wherein the T cells each have antigenic specificity for a human papillomavirus (HPV) 6 or HPV 11 antigen, the method comprising:culturing one or more fragments of the papilloma of the mammal ex vivo in the presence of one or more cytokines, wherein the papilloma is infected with HPV 6 or HPV 11 ;obtaining T cells from the cultured one or more fragments;inducing antigen presenting cells (APCs) of the mammal ex vivo to present one or more HPV 6 antigens or one or more HPV 11 antigens; andstimulating, for 10 to 19 days and in the presence of one or more cytokines ex vivo, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 6 antigens or the one or more HPV 11 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 6 or HPV 11 antigen.

8. The method of claim 7, wherein:the papilloma is infected with HPV 6,the method comprises inducing APCs of the mammal to present one or more HPV 6 antigens; andthe method comprises stimulating, for 10 to 19 days and in the presence of one or more cytokines ex vivo, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 6 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 6 antigen.

9. The method of claim 7, w herein:the papilloma is infected with HPV 11,the method comprises inducing APCs of the mammal to present one or more HPV 11 antigens; andthe method comprises stimulating, for 10 to 19 days and in the presence of one or more cytokines ex vivo, T cells from the cultured one or more fragments with the APCs that present the one or more HPV 11 antigens, thereby selectively expanding the number of T cells each having antigenic specificity for the HPV 11 antigen.

10. The method of any one of claims 7-9, wherein the method selectively expands the number of T cells each having antigenic specificity7for the HPV 6 antigen or the HPV 11 antigen by 20 to 40 fold.

11. The method of any one of claims 1-10, wherein the mammal has a condition caused by a chronic HPV 6 infection.

12. The method of any one of claims 1-10, wherein the mammal has a condition caused by a chronic HPV 11 infection.

13. The method of claim 11 or 12, wherein the condition is recurrent respiratory papillomatosis (RRP) or laryngotracheal disease.

14. The method of claim 11 or 12, wherein the condition is anogenital condyloma.

15. The method of any one of claims 11-14, wherein the condition is not cancerous and is not a pre-malignancy.

16. The method of any one of claims 11-14, wherein the condition is cancerous.

17. The method of any one of claims 1-16, wherein the T cells prepared by the method are ohgoclonal.

18. The method of any one of claims 1-17, wherein the T cells prepared by the method comprise multiple T cells, each T cell having antigenic specificity for a different HPV 6 antigen or a different HPV 11 antigen.

19. The method of any one of claims 1-18, wherein the T cells prepared by the method comprise multiple T cells, each T cell having antigenic specificity for the HPV 6 antigen or the HPV 11 antigen presented by a different human leukocyte antigen (HLA) element expressed by the mammal.

20. A method of isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen, the method comprising:preparing a T cell product from a papilloma from a mammal according to the method any one of claims 1-6 and 11-19; andisolating a TCR, or an antigen-binding portion thereof, from the expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for an HPV 6 or HPV 11 antigen.

21. A method of isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity an HPV 6 or HPV 11 antigen, the method comprising:selectively expanding a number of T cells from a papilloma of a mammal according to the method any one of claims 7-19; andisolating a TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for an HPV 6 or HPV 11 antigen.

22. A method of preparing a population of cells that express a TCR, or an antigenbinding portion thereof, having antigenic specificity for an HPV 6 or HPV 11 antigen, the method comprising:isolating a TCR, or an antigen-binding portion thereof, according to the method of claim 20 or 21, andintroducing the nucleotide sequence encoding the isolated TCR, or the antigenbinding portion thereof, into peripheral blood mononuclear cells (PBMC) ex vivo to obtain cells that express the TCR, or the antigen-binding portion thereof.23.A selectively expanded number of T cells prepared according to the method of any one of claims 7-19 for use in treating or preventing a chronic viral infection in a mammal, wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

24. A T cell product prepared according to the method of any one of claims 1-6 and 11-19 for use in treating or preventing a chronic viral infection in a mammal,wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

25. A population of cells that express a TCR, or an antigen-binding portion thereof, prepared according to the method of claim 22for use in treating or preventing a chronic viral infection in the mammal,wherein the chronic viral infection is a chronic HPV 6 infection or a chronic HPV 11 infection.

26. The selectively expanded number of T cells for the use of claim 23, the T cell product for the use of claim 24, or the population of cells that express a TCR, or an antigenbinding portion thereof, for the use of claim 25, wherein the condition caused by the chronic viral infection is recurrent respiratory papillomatosis (RRP) or laryngotracheal disease.

27. The selectively expanded number of T cells for the use of claim 23, the T cell product for the use of claim 24, or the population of cells that express a TCR, or an antigenbinding portion thereof, for the use of claim 25, wherein the condition caused by the chronic viral infection is anogenital condyloma.

28. The selectively expanded number of T cells for the use of claim 23, 26, or 27, the T cell product for the use of claim 24, 26, or 27, or the population of cells that express a TCR, or an antigen-binding portion thereof, for the use of any one of claims 25-27, wherein the mammal has a chronic HPV 6 infection.

29. The selectively expanded number of T cells for the use of claim 23, 26, or 27, the T cell product for the use of claim 24, 26, or 27, or the population of cells that express a TCR, or an antigen-binding portion thereof, for the use of any one of claims 25-27, wherein the mammal has a chronic HPV 11 infection.

30. The selectively expanded number of T cells for the use of any one of claims 23 and 26-29, the T cell product for the use of any one of claims 24 and 26-29, or the population of cells that express a TCR, or an antigen-binding portion thereof, for the use of any one of claims 25-29, further comprising one or more of a vaccine against HPV 6 or HPV 11, an antiprogrammed death-ligand (PD-L1) antibody, an anti-PD-1 antibody, and an anti-vascular endothelial growth factor inhibitor.

31. The selectively expanded number of T cells for the use of any one of claims 23 and 26-30, the T cell product for the use of any one of claims 24 and 26-30, or the population of cells that express a TCR, or an antigen-binding portion thereof, for the use of any one of claims 25-30, further comprising nonmyeloablative lymphodepleting chemotherapy.

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