Patent Application
20240076343
The instant application contains a Sequence Listing which is being submitted herewith electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jun. 5, 2023, is named 102085.021706_Sequence Listing.xml and is 696,000 bytes in size.
Disclosed herein are modified human interleukin-2 (hIL-2) proteins, human antibody molecules, or antigen-binding fragments thereof, that immunospecifically bind to human programmed cell death protein-1 (hPD-1), and immunoconjugates comprising the same.
Human IL-2 (hIL-2) is a Type 1 four α-helical bundle, glycosylated cytokine produced by CD4+ T cells and CD8+ T cells. Autocrine and paracrine IL-2 signaling occurs through engagement of either a high-affinity trimeric receptor complex comprising IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), or an intermediate-affinity dimeric receptor complex which comprises IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 has dual opposing and pleiotropic roles, in that it can both stimulate T cell proliferation to generate T cell effector, T cell memory, and activated NK cells, but can also stimulate suppressive regulatory T cells for maintenance of immune homeostasis. Low-dose IL-2 primarily stimulates regulatory T cells as well as some T effector and NK cells, whereas high-dose IL-2 broadly stimulates cytotoxic T cells, T effector, and NK cells and regulatory T cells. The use of IL-2 in the treatment of autoimmune diseases and as a cancer immunotherapy has, however, been limited by off-target effects and toxicity associated with the administration of IL-2.
Disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345, wherein the modified hIL-2 protein exhibits reduced potency on both a high affinity hIL-2 receptor and on an intermediate affinity hIL-2 receptor relative to the non-modified hIL-2.
Also disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20 and a R38E substitution at amino acid position 38 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345.
Also disclosed herein are human antibody molecules, or antigen-binding fragments thereof, that immunospecifically bind to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or antigen-binding fragment thereof comprises:
Also disclosed herein are immunoconjugates comprising:
Pharmaceutical compositions comprising any of the herein disclosed modified hIL-2 proteins, human antibody molecules, or antigen-binding fragments thereof, or immunoconjugates are also disclosed.
Also disclosed herein are polynucleotides comprising a nucleic acid sequence encoding any of the herein disclosed modified hIL-2 proteins, human antibody molecules, or antigen-binding fragments thereof, or immunoconjugates, as well as vectors comprising the polynucleotides and transformed cells comprising the vectors.
Disclosed herein are methods of treating a disease or disorder in a subject, the methods comprising administering a therapeutically effective amount of any of the herein disclosed immunoconjugates or pharmaceutical compositions to the subject to thereby treat the disease or disorder.
Also disclosed are uses of any of the herein disclosed immunoconjugates or pharmaceutical compositions in the preparation of a medicament for the treatment of a disease, and uses of any of the herein disclosed immunoconjugates or pharmaceutical compositions for the treatment of a disease or disorder.
The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates, there are shown in the drawings exemplary embodiments of the modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates; however, the modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates are not limited to the specific embodiments disclosed. In the drawings:
The disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates are not limited to the specific modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates.
Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
Throughout this text, the descriptions refer to modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates, as well as methods of using the modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates. Where the disclosure describes or claims a feature or embodiment associated with a modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate, such a feature or embodiment is equally applicable to the methods of using the modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate, such a feature or embodiment is equally applicable to the modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates.
Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the herein disclosure. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.
When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ±10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value.
It is to be appreciated that certain features of the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
As used herein, the singular forms “a,” “an,” and “the” include the plural.
Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.”
The term “antibody molecule” is meant in a broad sense and includes full length immunoglobulin molecules and antigen-binding fragments thereof.
Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
“Antigen-binding fragment” refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody (i.e., “antigen-binding fragment thereof”). Exemplary antigen binding fragments can have: heavy chain complementarity determining regions (CDR) 1, 2, and/or 3; light chain CDR 1, 2, and/or 3; a heavy chain variable region (VH); a light chain variable region (VL); and combinations thereof. Antigen binding fragments include: a Fab fragment, a monovalent fragment consisting of the VL, VH, constant light (CL), and constant heavy 1 (CH1) domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain or a VL domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody, described for example in Int'l Pat. Pub. Nos. WO1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.
The phrase “immunospecifically binds” refers to the ability of the disclosed antibody molecules to preferentially bind to its target (hPD-1 in the case of anti-hPD-1 antibody molecules) without preferentially binding other molecules in a sample containing a mixed population of molecules. Antibody molecules that immunospecifically bind hPD-1 are substantially free of other antibodies having different antigenic specificities (e.g., an anti-hPD-1 antibody is substantially free of antibodies that specifically bind antigens other than hPD-1). Antibody molecules that immunospecifically bind hPD-1, however, can have cross-reactivity to other antigens, such as orthologs of hPD-1, including Macaca fascicularis (cynomolgus monkey) PD-1. The antibody molecules disclosed herein are able to immunospecifically bind both naturally-produced hPD-1 and to PD-1 which is recombinantly produced in mammalian or prokaryotic cells.
An antibody variable region consists of four “framework” regions interrupted by three “antigen binding sites.” The antigen binding sites are defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); and (ii) “Hypervariable regions” (“HVR” or “HV”), three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of the antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The AbM definition of CDRs is also widely used; it is a compromise between Kabat and Chothia numbering schemes and is so-called because it was used by Oxford Molecular's AbM antibody modelling software (Rees, A. R., Searle, S. M. J., Henry, A. H. and Pedersen, J. T. (1996) In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). Other terms include “IMGT-CDRs” (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and “Specificity Determining Residue Usage” (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.
“Framework” or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.
“Human antibody,” “fully human antibody,” and like terms refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy and/or light chain variable regions that are “derived from” sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or chicken carrying human immunoglobulin loci as described herein. “Human antibody” may contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to, for example, naturally occurring somatic mutations or intentional introduction of substitutions in the variable domain (framework and antigen binding sites), or constant domain. Typically, a “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, as described in, for example, Shi et al., J Mol Biol 397:385-96, 2010 and Int'l Pat. Pub. No. WO2009/085462. Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of “human antibody.”
Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties in the variable regions or the constant regions or both, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.
“Recombinant antibody” includes all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as: antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below); antibodies isolated from a host cell transformed to express the antibody; antibodies isolated from a recombinant, combinatorial antibody library; and antibodies prepared, expressed, created, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange.
“Monoclonal antibody” refers to a population of antibody molecules of a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes. Monoclonal antibody therefore refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
“Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar, or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.
“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions, or deletions. The term “mutation” as used herein is intended to mean one or more intentional substitutions which are made to a polypeptide or polynucleotide.
“Treat,” “treatment,” and like terms refer to both therapeutic treatment and prophylactic or preventative measures, and includes reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause of the symptoms, reducing the frequency or likelihood of symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by the disease or disorder. Treatment also includes prolonging survival as compared to the expected survival of a subject not receiving treatment. Subjects to be treated include those that have the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented.
As used herein, “administering to the subject” and similar terms indicate a procedure by which the disclosed modified hIL-2 proteins, immunoconjugates, or pharmaceutical compositions are injected into a subject such that target cells, tissues, or segments of the body of the subject are contacted with the disclosed modified hIL-2 proteins or immunoconjugates comprising the same.
The phrase “therapeutically effective amount” refers to an amount of the modified hIL-2 proteins, immunoconjugates, or pharmaceutical compositions, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the modified hIL-2 proteins, immunoconjugates, or pharmaceutical compositions to cause a desired response in a subject. Exemplary indicators of a therapeutically effect amount include, for example, improved well-being of the patient, reduction of a disease symptom, arrested or slowed progression of disease symptoms, and/or absence of disease symptoms.
The term “subject” as used herein is intended to mean any animal, in particular, mammals. Thus, the methods are applicable to human and nonhuman animals, although most preferably used with humans. “Subject” and “patient” are used interchangeably herein.
Immunoconjugate and fusion protein are used interchangeably herein.
Modified Human Interleukin-2 (hIL-2) Proteins
Disclosed herein are modified hIL-2 proteins comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345, wherein the modified hIL-2 protein exhibits reduced potency on both a high affinity hIL-2 receptor and on an intermediate affinity hIL-2 receptor relative to a non-modified hIL-2. The disclosed modified hIL-2 proteins are also referred to as “attenuated” hIL-2 herein.
Suitable substitutions at amino acid position 20 include, for example, any one of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution.
Suitable substitutions at amino acid position 38 include, for example, any one of an R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or an R38K substitution.
In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with an R38E substitution.
The modified hIL-2 proteins can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 proteins can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 can further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.
The modified hIL-2 proteins may comprise a D20A substitution and a R38E substitution.
As described herein the term “reduced potency” and related terms such as “reduction in potency” or “attenuation” of IL-2 activity refer to a reduction in potency of the modified hIL-2 as determined by an increased EC50 value relative to the EC50 value for an non-modified-hIL-2 in an IL-2-dependent assay. As described herein the reduction in potency of the modified hIL-2 will be on both the high affinity and on the intermediate affinity IL-2 receptors. The IL-2-dependent assay for determining potency may be an engineered human erythroleukemic TF1 (TF1+IL-2Rβ) or a human natural killer NK-92 cell proliferation assay as described herein. In one embodiment, the IL-2-dependent assay for determining potency is an engineered human erythroleukemic TF1 (TF1+IL-2Rβ) cell proliferation assay. In another embodiment, the IL-2-dependent assay for determining potency is a human natural killer NK-92 cell proliferation assay. Other IL-2-dependent assays for determining potency may also be a TF1+IL-2Rβ or a human natural killer NK-92 pSTAT5 assay as described herein. The non-modified-hIL-2 may be a prokaryote-expressed hIL-2 such as Proleukin® (which has the native human IL-2 amino acid sequence apart from a C125S substitution to remove an unbound cysteine, and which does not bear the normal human carbohydrate expression on residue T3), or the non-modified-hIL-2 may be an hIL-2 with the amino acid sequence of SEQ ID NO: 345 or with the amino acid sequence of SEQ ID NO: 345 with a C125S substitution, which is expressed in a mammalian cell line, such as a CHO or HEK cell line.
The modified hIL-2 proteins can further comprise a substitution at amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. A suitable substitution includes, for example, a T3A. In some embodiments, the modified hIL-2 proteins comprise a T3A substitution, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 216.
Alternatively, the modified hIL-2 proteins can further comprise a deletion of amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. In some embodiments, the modified hIL-2 proteins comprise a deletion of amino acids 1-3, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 218.
The modified hIL-2 proteins can further comprise a deletion or substitution at amino acid position 125 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. The substitution at amino acid position 125 can be C125A. In some embodiments, the modified hIL-2 proteins comprise a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 proteins comprise a T3A substitution, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 proteins comprise a deletion of amino acids 1-3, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 219.
The modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2. A greater reduction in hIL-2 potency on the high affinity hIL-2 receptor may be possible and acceptable for the modified hIL-2 proteins described herein, but such a reduction may not be quantifiable with the methods described herein due to limits of the cell proliferation assay conditions.
In addition, the modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2.
The modified hIL-2 proteins can exhibit a reduction in potency of up to about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of up to about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. The modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2
As demonstrated herein, the modified hIL-2 proteins can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof. The hIL-2 proteins can fused to the anti-PD-1 antibody or an antigen-binding fragment thereof at the N-terminus of an antibody light chain, the C-terminus of an antibody light chain, the N-terminus of an antibody heavy chain, the C-terminus of an antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein is directly fused by a peptide bond to the anti-PD-1 antibody or an antigen-binding fragment thereof. The modified hIL-2 proteins can be, for example, directly fused by a peptide bond to the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain. In some embodiments, the modified hIL-2 protein is fused to the anti-PD-1 antibody or an antigen-binding fragment thereof through a linker.
Fusion of the modified hIL-2 proteins to the antibody or antigen-binding fragments thereof can rescue the modified hIL-2 proteins' ability to bind to and activate the human intermediate affinity IL-2 receptor on PD-1-expressing cells such as T cells and in particular tumor-infiltrating lymphocytes. In some embodiments, the hIL-2 protein that is fused to the antibody or an antigen-binding fragment thereof exhibits potency on the intermediate affinity IL-2 receptor on PD-1-expressing cells that is comparable to the potency of wild type hIL-2 on the intermediate affinity IL-2 receptor.
Fusion of the modified hIL-2 protein to an antibody or antigen-binding fragment thereof can be used to selectively deliver IL-2 signaling to cells expressing the PD-1 target of the antibody or antigen-binding fragment thereof. Without being bound by theory, it is believed that targeting the modified hIL-2 protein to specific cell populations can dramatically amplify the therapeutic effects of the IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicities.
Also disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20 and a R38E substitution at amino acid position 38 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345.
The modified hIL-2 proteins can comprise the amino acid sequence of any one of SEQ ID NOs: 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 307, 607-611, 614, 617, or 620 can further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 307, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 307, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 307, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.
The modified hIL-2 proteins may comprise a D20A substitution and a R38E substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 149.
The modified hIL-2 proteins can further comprise a substitution at amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. A suitable substitution includes, for example, a T3A. In some embodiments, the modified hIL-2 proteins comprise a T3A substitution, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 216.
Alternatively, the modified hIL-2 proteins can further comprise a deletion of amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. In some embodiments, the modified hIL-2 proteins comprise a deletion of amino acids 1-3, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 218.
The modified hIL-2 proteins can further comprise a deletion or substitution at amino acid position 125 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. The substitution at amino acid position 125 can be C125A. In some embodiments, the modified hIL-2 proteins comprise a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 proteins comprise a T3A substitution, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 proteins comprise a deletion of amino acids 1-3, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 219.
The modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2. A greater reduction in hIL-2 potency on the high affinity hIL-2 receptor may be possible and acceptable for the modified hIL-2 proteins described herein, but such a reduction may not be quantifiable with the methods described herein due to limits of the cell proliferation assay conditions.
In addition, the modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2.
The modified hIL-2 proteins can exhibit a reduction in potency of up to about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of up to about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. The modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2.
As demonstrated herein, the modified hIL-2 proteins can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof. The hIL-2 proteins can fused to the anti-PD-1 antibody or an antigen-binding fragment thereof at the N-terminus of an antibody light chain, the C-terminus of an antibody light chain, the N-terminus of an antibody heavy chain, the C-terminus of an antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein is directly fused by a peptide bond to the anti-PD-1 antibody or an antigen-binding fragment thereof. The modified hIL-2 proteins can be, for example, directly fused by a peptide bond to the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain. In some embodiments, the modified hIL-2 protein is fused to the anti-PD-1 antibody or an antigen-binding fragment thereof through a linker.
Fusion of the modified hIL-2 proteins to the antibody or antigen-binding fragments thereof can rescue the modified hIL-2 proteins' ability to bind to and activate the human intermediate affinity IL-2 receptor on PD-1-expressing cells such as T cells and in particular tumor-infiltrating lymphocytes. In some embodiments, the hIL-2 protein that is fused to the antibody or an antigen-binding fragment thereof exhibits potency on the intermediate affinity IL-2 receptor on PD-1-expressing cells that is comparable to the potency of wild type hIL-2 on the intermediate affinity IL-2 receptor.
Fusion of the modified hIL-2 protein to an antibody or antigen-binding fragment thereof can be used to selectively deliver IL-2 signaling to cells expressing the PD-1 target of the antibody or antigen-binding fragment thereof. Without being bound by theory, it is believed that targeting the modified hIL-2 protein to specific cell populations can dramatically amplify the therapeutic effects of the IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicities.
Human Anti-Human Programmed Cell Death Protein-1 (hPD-1) Antibodies
Disclosed herein are human antibody molecules, or antigen-binding fragments thereof, that immunospecifically bind to hPD-1, wherein the human antibody molecule or antigen-binding fragment thereof comprises:
In some embodiments, the human antibody molecules, or antigen-binding fragments thereof, comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423 (referred to herein as “H7-632”).
In some embodiments, the human antibody molecules, or antigen-binding fragments thereof, comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391 (referred to herein as “2H7”).
In some embodiments, the human antibody molecules, or antigen-binding fragments thereof, comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 401 (referred to herein as “C51E6-5”).
In some embodiments, the human antibody molecules, or antigen-binding fragments thereof, comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 411 (referred to herein as “A2”).
The disclosed human antibody molecules or antigen-binding fragments thereof, can exhibit one or more of the following activities:
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417 (referred to herein as “H7-632”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385 (referred to herein as “2H7”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395 (referred to herein as “C51E6-5”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 405 (referred to herein as “A2”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a human IgG1 heavy chain constant region.
The human antibody molecules, or antigen binding fragments thereof, can have substitutions or deletions within the constant region to minimize Fc-mediated immune effector function, such as FcγRIIIA-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRI- and FcγRIIa-dependent antibody-dependent cellular phagocytosis (ADCP), and C1q binding-mediated complement-dependent cytotoxicity (CDC). In some embodiments, the human antibody molecules comprise a L235A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecules comprise a G237A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecules comprise an L235A and a G237A substitution, wherein the amino acid numbering is according to EU numbering.
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415 (referred to herein as “H7-632-hIgG1-LAGA”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425 (referred to herein as “2H7-hIgG4”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427 (referred to herein as “C51E6-5-hIgG4”).
The human antibody molecules, or antigen-binding fragments thereof, can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 428 and a light chain comprising the amino acid sequence of SEQ ID NO: 429 (referred to herein as “A2-hIgG4”).
The human antibody molecules, or antigen-binding fragments thereof, can be fused to a modified hIL-2 protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. The human antibody molecule, or antigen-binding fragments thereof, can be fused to any of the herein disclosed modified hIL-2 proteins.
When not fused to the antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit reduced potency on both a high affinity hIL-2 receptor and on an intermediate affinity hIL-2 receptor relative to a non-modified hIL-2.
Suitable substitutions at amino acid position 20 of the modified hIL-2 include, for example, any one of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution.
Suitable substitutions at amino acid position 38 of the modified hIL-2 protein include, for example, any one of an R38E, R38N, R38G, R38H, R381, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or a R38K substitution.
In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with an R38E substitution.
The modified hIL-2 proteins can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 proteins can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 can further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.
The modified hIL-2 protein can comprise a D20A substitution and a R38E substitution.
The modified hIL-2 protein can further comprise a substitution at amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. A suitable substitution includes, for example, a T3A. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 216.
Alternatively, the modified hIL-2 protein can further comprise a deletion of amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.
The modified hIL-2 protein can further comprise a deletion or substitution at amino acid position 125 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. The substitution at amino acid position 125 can be C125A. In some embodiments, the modified hIL-2 protein comprises a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 219.
When not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, when not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2. A greater reduction in hIL-2 potency on the high affinity hIL-2 receptor may be possible and acceptable for the modified hIL-2 proteins described herein, but such a reduction may not be quantifiable with the methods described herein due to limits of the cell proliferation assay conditions.
In addition, when not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, when not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2.
When not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of up to about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of up to about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. When not fused to the human antibody molecules or antigen-binding fragments thereof, the modified hIL-2 proteins can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2.
Fusion of the modified hIL-2 proteins to the antibody or antigen-binding fragments thereof can rescue the modified hIL-2 proteins' ability to bind to and activate the human intermediate affinity IL-2 receptor on PD-1-expressing cells such as T cells and in particular tumor-infiltrating lymphocytes. In some embodiments, the hIL-2 protein that is fused to the antibody or an antigen-binding fragment thereof exhibits potency on the intermediate affinity IL-2 receptor on PD-1-expressing cells that is comparable to the potency of wild type hIL-2 on the intermediate affinity IL-2 receptor
The modified hIL-2 proteins can be fused to the human antibody molecules or antigen-binding fragments thereof at the N-terminus of an antibody light chain, the C-terminus of an antibody light chain, the N-terminus of an antibody heavy chain, the C-terminus of an antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein is directly fused by a peptide bond to the antibody or an antigen-binding fragment thereof. The hIL-2 can be, for example, directly fused by a peptide bond to the C-terminal amino acid residue of the antibody heavy chain. In some embodiments, the hIL-2 protein is fused to the antibody or an antigen-binding fragment thereof through a linker.
Fusion of the human antibody molecules or antigen-binding fragments thereof to the modified hIL-2 proteins can be used to selectively deliver IL-2 signaling to cells expressing PD-1. Without being bound by theory, it is believed that targeting the modified hIL-2 protein to specific cell populations expressing PD-1 can dramatically amplify the therapeutic effects of the IL-2 (e.g., anti-tumor immunity) while reducing or minimizing off-target systemic toxicities.
Disclosed herein are immunoconjugates comprising any of the herein disclosed modified hIL-2 proteins and any of the herein disclosed human antibody molecules, or antigen-binding fragments thereof. The immunoconjugates can comprise:
Suitable substitutions at amino acid position 20 of the modified hIL-2 portion of the immunoconjugates include, for example, any of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution.
Suitable substitutions at amino acid position 38 of the modified hIL-2 portion of the immunoconjugates include, for example, any of an R38E, R38N, R38G, R38H, R381, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or a R38K substitution.
In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with an R38E substitution.
The modified hIL-2 protein portion of the immunoconjugates can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein portion of the immunoconjugates can comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 proteins comprise the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 can further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of amino acid sequences SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.
The modified hIL-2 protein portion of the immunoconjugates can comprise a D20A substitution and a R38E substitution.
The modified hIL-2 protein portion of the immunoconjugates can further comprise a substitution at amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. A suitable substitution includes, for example, a T3A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise a T3A substitution, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugates comprise the amino acid sequence of SEQ ID NO: 216.
Alternatively, the modified hIL-2 protein portion of the immunoconjugates can further comprise a deletion of amino acid position 3 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise a deletion of amino acids 1-3, a D20A substitution, and a R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugates comprise the amino acid sequence of SEQ ID NO: 218.
The modified hIL-2 protein portion of the immunoconjugates can further comprise a deletion or substitution at amino acid position 125 relative to the non-modified hIL-2 amino acid sequence of SEQ ID NO: 345. The substitution at amino acid position 125 can be C125A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise a T3A substitution, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise a deletion of amino acids 1-3, a D20A substitution, a R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates comprise the amino acid sequence of SEQ ID NO: 219.
The modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) relative to a non-modified hIL-2. A greater reduction in hIL-2 potency on the high affinity hIL-2 receptor may be possible and acceptable for the modified hIL-2 proteins described herein, but such a reduction may not be quantifiable with the methods described herein due to limits of the cell proliferation assay conditions.
In addition, the modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to an non-modified hIL-2.
The modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of up to about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of up to about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2, for example as quantified by a comparison in EC50 values in an hIL-2-dependent cell proliferation assay described herein. The modified hIL-2 protein portion of the immunoconjugates can exhibit a reduction in potency of greater than about 10,000-fold on the high affinity IL-2 receptor (hIL-2Rαβγ) and a reduction in potency of greater than about 10,000-fold on the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to a non-modified hIL-2.
The hIL-2 protein portion of the immunoconjugates can be fused to the antibody or an antigen-binding fragment thereof at the N-terminus of an antibody light chain, the C-terminus of an antibody light chain, the N-terminus of an antibody heavy chain, the C-terminus of an antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein portion of the immunoconjugates is directly fused by a peptide bond to the human antibody molecule or an antigen-binding fragment thereof. The hIL-2 protein portion of the immunoconjugates can be, for example, directly fused by a peptide bond to the C-terminal amino acid residue of the antibody heavy chain. In some embodiments, the hIL-2 protein portion of the immunoconjugates is fused to the human antibody molecule or an antigen-binding fragment thereof through a linker.
Fusion of the modified hIL-2 proteins to the human antibody molecules or antigen-binding fragments thereof can rescue the modified hIL-2 proteins' ability to activate the intermediate affinity IL-2 receptor. In some embodiments, the immunoconjugate is able to activate the intermediate affinity IL-2 receptor to a degree that is comparable to wild type hIL-2 activation of the intermediate affinity IL-2 receptor.
In some embodiments, the human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423.
In some embodiments, the human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391.
In some embodiments, the human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 396, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 397, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 398, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 399, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 400, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 401.
In some embodiments, the human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 411.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 405.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise an IgG1 heavy chain constant region.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can have substitutions or deletions within the constant region to minimize Fc-mediated immune effector function, such as FcγRIIIA-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRI- and FcγRIIa-dependent antibody-dependent cellular phagocytosis (ADCP), and C1q binding-mediated complement-dependent cytotoxicity (CDC). In some embodiments, the human antibody molecule portion of the immunoconjugates comprise a L235A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecule portion of the immunoconjugates comprise a G237A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecule portion of the immunoconjugates comprise an L235A and a G237A substitution, wherein the amino acid numbering is according to EU numbering.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427.
The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugates can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 428 and a light chain comprising the amino acid sequence of SEQ ID NO: 429.
The immunoconjugates can have one or more of the following properties:
In some embodiments, the immunoconjugate comprises a modified hIL-2 protein comprising a T3A substitution, a R38E substitution, a D20A substitution, and a C125A substitution fused to the C-terminus of the antibody heavy chain of a human anti-hPD-1 antibody comprising a human IgG1 framework with a L235A substitution and a G237A substitution. In some embodiments, the immunoconjugate comprises a light chain comprising the amino acid sequence of SEQ ID NO: 415 and a heavy chain-hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO: 532.
The disclosed immunoconjugates can selectively deliver IL-2 signaling to PD-1-expressing T cells. The human antibody molecule, or antigen-binding fragment thereof, portion of the immunoconjugate is utilized solely to deliver the modified hIL-2 to PD-1-expressing cells and does not block PD-1 receptor function, as do classical anti-PD-1 inhibitor antibodies such as OPDIVO® and KEYTRUDA®. The primary mechanism-of-action of the herein disclosed immunoconjugates is via the T cell selective activity of IL-2. The human PD-1 receptor is primarily expressed on a minor subset of T cells with potent tumor reactivity. Without being bound by theory, it is believed that targeting the modified hIL-2 protein portion of the immunoconjugate to this population of T cells can dramatically amplify anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicities mediated by cell populations that lack PD-1 expression.
Disclosed herein are pharmaceutical compositions comprising any of the herein disclosed modified hIL-2 proteins, any of the herein disclosed human antibody molecules or antigen-binding fragments thereof, or any of the herein disclosed immunoconjugates. In some embodiments, the pharmaceutical compositions comprise any of the herein disclosed modified hIL-2 proteins. In some embodiments, the pharmaceutical compositions comprise any of the herein disclosed human antibody molecules or antigen-binding fragments thereof. In some embodiments, the pharmaceutical compositions comprise any of the herein disclosed immunoconjugates.
Disclosed herein are polynucleotides comprising a nucleic acid sequence encoding any of the herein disclosed modified hIL-2 proteins, any of the herein disclosed human antibody molecules or antigen-binding fragments thereof, or any of the herein disclosed immunoconjugates. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding any of the herein disclosed modified hIL-2 proteins. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding any of the herein disclosed human antibody molecules or antigen-binding fragments thereof. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding any of the herein disclosed immunoconjugates.
Disclosed herein are vectors comprising a polynucleotide comprising a nucleic acid sequence that encodes any of the herein disclosed modified hIL-2 proteins, any of the herein disclosed human antibody molecules or antigen-binding fragments thereof, or any of the herein disclosed immunoconjugates. In some embodiments, the vectors comprise a polynucleotide comprising a nucleic acid sequence that encodes any of the herein disclosed modified hIL-2 proteins. In some embodiments, the vectors comprise a polynucleotide comprising a nucleic acid sequence that encodes any of the herein disclosed human antibody molecules or antigen-binding fragments thereof. In some embodiments, the vectors comprise a polynucleotide comprising a nucleic acid sequence that encodes any of the herein disclosed immunoconjugates.
Also disclosed herein are transformed cells comprising any of the herein disclosed vectors.
Disclosed herein are methods of treating a disease or disorder in a subject, the methods comprising administering a therapeutically effective amount of any of the herein disclosed immunoconjugates or pharmaceutical compositions to the subject to thereby treat the disease.
Also disclosed are uses of any of the herein disclosed immunoconjugates or pharmaceutical compositions in the preparation of a medicament for the treatment of a disease. Also disclosed are uses of any of the herein described immunoconjugates or pharmaceutical compositions for the treatment of a disease or disorder.
The disclosed immunoconjugates and pharmaceutical compositions can be used to treat diseases or disorders in which stimulation of the subject's immune system would be beneficial. In some embodiments, the subject has an insufficient or deficient immune response and the disclosed immunoconjugates and pharmaceutical compositions stimulate the subject's immune response. The antibody portion of the immunoconjugate can serve to direct the modified hIL-2 protein to the subject's immune cells by, for example, binding to an antigen expressed on the surface of the immune cell. In the case of the disclosed modified hIL-2 protein-human anti-hPD-1 antibody immunoconjugates, for example, the anti-PD-1 antibody (or antigen-binding fragment thereof) portion of the immunoconjugate can bind PD-1 expressed on T cells, thereby delivering the modified hIL-2 protein to the T cells. Targeting the modified IL-2 protein to specific cells can dramatically amplify the therapeutic efficacy of the IL-2 protein without off-target systemic toxicities mediated by cell populations that lack the antigen expression. The disclosed methods and uses can be used to treat, for example, cancer, autoimmune diseases and inflammatory diseases, and chronic infections and infectious diseases. Exemplary cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, non-small cell lung carcinoma, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, melanoma, squamous cell carcinoma, bone cancer, and kidney cancer. Exemplary autoimmune diseases and inflammatory disease include systemic lupus erythematosus (SLE), Type 1 diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriasis, Behcet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, sclerosing cholangitis, Sjoren's syndrome, alopecia areata, and inflammatory myopathies. Exemplary infectious diseases include HIV and hepatitis B.
In some embodiments, the disease is cancer. The methods and uses can comprise administering a therapeutically effective amount of any of the herein disclosed modified hIL-2 protein-antibody conjugates to the subject to thereby treat the cancer. In some aspects, the cancer is melanoma. In some aspects, the cancer is non-small cell lung carcinoma.
The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Protocol A. Flow Cytometry Screen for Binding of Anti-hPD-1 Antibodies or Anti-hPD-1 Antibody-Attenuated hIL-2 Fusions to Human PD-1
To test for binding to hPD-1, antibodies and antibody-attenuated hIL-2 fusion proteins were characterized in full titration curves. A Jurkat cell line was transfected with a mammalian vector which encoded amino acids 1-185 of human PD-1 (SEQ ID NO: 346) to stably express the extracellular domain and a portion of the transmembrane domain of human PD-1, and this transfected cell line was used to determine binding of anti-hPD-1 antibodies. Jurkat+hPD-1 cells were washed and added to 96-well plates at 100,000 cells per well in FACS buffer (PBS, 0.2% Heat-inactivated Fetal Bovine Serum). Cells were blocked with 1:50 dilution of human FcR Block (Miltenyi) for 10 minutes at 4° C. and washed with FACS buffer.
Antibodies or antibody-attenuated hIL-2 immunoconjugates (fusion proteins) were serially diluted six-fold in FACS buffer for an 8-point curve and added to human PD-1 expressing Jurkat cells for 1 hour on ice in 100 μL volume. Cells were washed and re-suspended in FACS buffer containing 1:40 dilution of Allophycocyanin conjugated anti-human IgG Fc monoclonal antibody. Cells were washed once more, re-suspended in FACS buffer containing 1:1000 dilution of Sytox Green (Thermo Fisher) and flow cytometric analysis was conducted on the BD FACS Canto II, BD Celesta or BD Fortessa (BD Biosciences) flow cytometers. The geometric mean fluorescent intensity (gMFI) was calculated using FlowJo software version 10. Half maximal effective concentration (EC50) values were calculated from the gMFI of the Allophycocyanin signal across the titrated concentrations using GraphPad Prism 7 software.
Protocol B. Flow Cytometry Competition Screen for Binding of Anti-hPD-1 Antibodies or Anti-hPD-1 Antibody-Attenuated hIL-2 Fusions to Human PD-1
Antibodies and antibody-attenuated hIL-2 fusion proteins were tested for the ability to bind human PD-1 in the presence of a saturating concentration of anti-hPD-1 #1-mIgG2b-N297A (sequence comprising the heavy and light chain variable region sequences of nivolumab, clone 5C4, as described in U.S. Patent Pub. No. US 2009/0217401A1, formatted onto a murine IgG2b-N297A background) (SEQ ID NOs: 348 and 349) or anti-hPD-1 #2-mIgG2b-N297A (sequence comprising the heavy and light chain variable region sequences of pembrolizumab (clone 109A-H/K09A-L-11) as described in Int'l Pub. No. WO2008/156712A1, formatted onto a murine IgG2b-N297A background) (SEQ ID NOs: 350 and 351).
Antibodies or antibody-attenuated hIL-2 fusion proteins were serially diluted six-fold for an 8-point titration curve with and without saturating amounts of 10 μM anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Briefly, Jurkat cells stably expressing hPD-1 (as described in Protocol A above) were washed and re-suspended in FACS buffer containing 1:50 dilution of human FcR Blocking reagent. Cells were incubated at 4° C. for 10 minutes and washed. Cells were then re-suspended in 100 μL volume with anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N287A diluted in FACS buffer to 10 μM and incubated at 4° C. for one hour. Cells were washed and incubated with test antibodies or antibody-attenuated hIL-2 fusion proteins serially diluted six-fold for an 8-point curve in 100 μL volume for one hour at 4° C. To detect bound test anti-hPD-1 antibodies or anti-hPD-1-attenuated hIL-2 fusion proteins, cells were washed again and incubated with 1:40 dilution of Allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody for 45 minutes on ice. Cells were washed and re-suspended in FACS buffer containing 1:1000 dilution of Sytox Green (Thermo Fisher). To generate a comparison, Jurkat cells stably expressing human PD-1 were incubated with only the titrated test antibodies or antibody-attenuated hIL-2 fusion proteins (without anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A) and subsequently with 1:40 dilution of Allophycocyanin-conjugated anti-human IgG Fc secondary. As a control, the variable regions of anti-hPD-1 #1 and anti-hPD-1 #2 were cloned into hIgG4 frameworks and were assessed with and without the addition of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Flow cytometry was carried out on the BD Canto II, BD Celesta, or BD Fortessa (BD Biosciences) flow cytometers and gMFI was calculated using FlowJo software version 10. EC50 values were calculated from the gMFI of the Allophycocyanin signal across the titrated concentrations using GraphPad Prism 7 software.
Protocol C. Cell-Based Screen for Characterization of Non-Antagonist Anti-hPD-1 Antibodies or Anti-hPD-1 Antibody-Attenuated hIL-2 Fusions
Human PD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were characterized for the ability to block hPD-1 from binding to ligand hPD-L1 (SEQ ID NO: 584). Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were either characterized as an antagonist or non-antagonist using an in vitro cell-based human PD-1/PD-L1 blockade bioassay (Promega, Cat #J1255). This co-culture assay utilized two cell lines: FCγR11b artificial Antigen Presenting Cells/Chinese Ovary Hamster K1 (aAPC/CHO-K1) and Jurkat Effector cells. aAPC/CHO K1 cells stably express both human PD-L1 ligand and a cell surface protein to activate cognate T cell receptors (TCRs) while Jurkat Effector cells express hPD-1 and a luciferase reporter under the control of Nuclear Factor of Activated T cells response element (NFAT-RE). When these cells are co-cultured in the presence of a non-antagonistic antibody, hPD-1/hPD-L1 interaction inhibits TCR signaling and no luminescence is detected. In the presence of an antibody that antagonizes hPD-1 interaction with hPD-L1 (SEQ ID NO: 584), the inhibitory signal is disrupted and luminescence is detected.
The thaw-and-use assay was performed according to manufacturer's instructions. In short, aAPC/CHO-K1 cells were first thawed and plated at 30,000 cells per well in flat-bottom 96-well plates for 18 hours at 37° C. in a 5% CO2 incubator. After cells had adhered, the media was removed and 200 nM or 1000 nM test antibodies or antibody-attenuated hIL-2 fusion proteins were diluted in 40 μL assay buffer (RPMI 1640 medium+1% FBS) and added to the aAPC/CHO-K1 cells. A human IgG4 isotype control monoclonal antibody which targeted Keyhole Limpet Hemocyanin (KLH) clone C3 (SEQ ID NOs: 585 and 586) was used as a negative control. Jurkat effector cells expressing hPD-1 were added at 24,000 cells per well in 40 μL volume. The final concentration of fixed antibodies tested was 100 nM or 500 nM. In some examples, a range of concentrations of anti-hPD-11 or anti-hPD-1-attenuated hIL-2 fusion proteins were tested in this co-culture assay, with the top concentration in a five-fold titration series of 500 nM (
The co-culture assay was incubated at 37° C. in a 5% CO2 incubator for an additional 18-20 hours. To read the luminescence signal, plates were allowed to come to room temperature, and 80 μL of the Bio-Glo™ reagent was added to each well. The plates were incubated for 15 minutes in the dark at room temperature and luminescence was read on a Victor X luminometer (Perkin Elmer). Relative luminescence units (RLU) were averaged for each triplicate and graphed using GraphPad Prism 7 software.
Protocol D. In Vitro Phosphorylated STAT5 Assay to Test Attenuation of hIL-2 Variants
The level of attenuation of hIL-2 receptor activation activity of antibody-attenuated hIL-2 fusion proteins was characterized using a phosphorylated STAT5 assay. Variants were tested in both hIL-2 responsive human natural killer NK-92 cells and engineered human erythroleukemic TF1 cells. The NK-92 cell line naturally expresses the high-affinity hIL-2 receptor (IL-2Rαβγ) at physiologic levels, while the TF1 cell line that naturally expresses the IL-2Rγ (SEQ ID NO: 352) was engineered to also stably express human CD122 (IL-2Rβ) (SEQ ID NO: 353) for expression of the intermediate affinity hIL-2 receptor complex (IL-2Rβγ). This TF1+IL-2Rβ stable cell line does not express the IL-2Rα (SEQ ID NO: 354). Both NK-92 and TF1+IL-2Rβ cell lines were used to assess the level of attenuation of IL-2 potency in these cell-based potency assays as fixed concentration screens and full titration curves.
To perform the fixed concentration screen, 100,000 NK-92 cells or TF1+IL-2Rβ cells were plated into 96 wells in 50 μL of fresh growth medium lacking human IL-2 cytokine and incubated overnight at 37° C. in a CO2 incubator. After 15-16 hours, human IL-2 starved cells were treated with 25.7 nM recombinant hIL-2 (denoted as rhIL-2) (SEQ ID NO: 345) or test antibody-attenuated hIL-2 fusion proteins for the NK-92 cell assay, or with 33.3 nM hIL-2 or test hIL-2 variants for the TF1+IL-2Rβ cell assay. Cells were incubated at 37° C., 5% CO2 for 10 minutes. Cells were fixed with Cytofix Buffer (BD Biosciences) for 10 minutes at 37° C. and then permeabilized after treatment with Perm Buffer III (BD Biosciences) for 30 minutes on ice. hIL-2-dependent Stat5 phosphorylation was detected after staining fixed and permeabilized cells with Alexa Fluor-647 conjugated anti-Stat5 antibody (BD Biosciences) at 0.5 μL per sample for 45 minutes at room temperature in the dark. Cells were washed and reagents were diluted in BD Pharmingen Buffer (BD Biosciences). Stained cells were acquired on a FACS-Celesta cytometer (BD Biosciences) and analyzed using FlowJo software version 10.7.2. The assays were performed in cohorts but normalized using the rhIL-2 for each plate. The degree of attenuation of selected antibody-attenuated hIL-2 fusion proteins were evaluated in an 8-point, 6-fold serially titrated curve ranging from 1200 nM to 7 μM on both NK-92 and TF1+IL-2Rβ cell lines. The procedure for the pStat5 curves was performed in the same manner as the method described above. EC50 values were calculated from the geometric mean fluorescent intensity (gMFI) across the titrated concentrations using GraphPad Prism 7 software. The fold change in activity from rhIL-2 was calculated by dividing the EC50 values for the variants by the EC50 of hIL-2.
Protocol E. In Vitro Cell-Based Proliferation Assay to Test Attenuation of Antibody-Attenuated hIL-2 Fusion Proteins
The antibody-attenuated hIL-2 fusion proteins were also tested for attenuated hIL-2 activity in hIL-2 dependent cell proliferation assays. 10,000 NK-92 cells (expressing the high affinity receptor hIL-2Rαβγ) or TF1+IL-2Rβ cells (expressing the intermediate affinity receptor hIL-2Rβγ) suspended in 50 μL of fresh growth medium without hIL-2 cytokine were plated per well in 96-well U-bottom cell culture plate. Eight point, 6-fold serial titrations of antibody-attenuated hIL-2 fusion proteins with a highest concentration of 996 nM were diluted in fresh media and overlaid on cells in wells. Cells were incubated at 37° C. in a 5% C02 incubator for 3 days for TF1+IL-2Rβ cells or 4 days for NK-92 cells. To measure proliferation, Cell-Titer-Glo (Promega) was added to wells, incubated for 10 minutes at room temperature and luminescence was read for 0.1 second per well using a VictorX Multilabel Plate Reader (Perkin Elmer). EC50 values were calculated from the relative luminescence units (RLU) across the titrated concentrations using GraphPad Prism 7 software. The fold change in activity from rhIL-2 was calculated by dividing the EC50 values for the variants by the EC50 of hIL-2. The assays were performed in cohorts but normalized using the rhIL-2 EC50 value for each plate.
In order to determine the optimal structures for an antibody-attenuated hIL-2 fusion protein, non-attenuated hIL-2 was fused to an anti-DNase I antibody (clone 1H3) designated as 1H3-hIgG1 (SEQ ID NO: 379, SEQ ID NO: 374) in the antibody variable region in a variety of ways as illustrated in
Table 1 summarizes the EC50 calculated over the 8-point, 6-fold serially titrated curves using the geometric mean fluorescence intensity (gMFI) calculated by the FlowJo version 10 software. The fold change from rhIL-2 was also calculated for each variant as a measurement of the level of attenuation as compared to the activity of the rhIL-2 positive control. Some EC50 values were unable to be calculated by the GraphPad Prism 7 software and were marked as Not Calculated (NC); however, based on dose-titration curves there was no attenuation for these variants.
Fusions of the hIL-2 moiety to the N-terminus or C-terminus of the immunoglobulin heavy chain resulted in no reduction in TL-2 activity when compared to rhIL-2 on cell lines expressing the high-affinity hIL-2 receptor (NK-92) or intermediate-affinity hIL-2 receptor (TF1+IL-2Rβ). The direct fusion (df) of hIL-2 to the antibody component of the fusion protein resulted in no change in TL-2 activity when compared with fusion employing a six amino acid linker (L6) between the IL-2 and antibody components. Similarly, fusions of the TL-2 component to the heavy chain or light chain of the antibody component resulted in no change in IL-2 activity when compared to rhIL-2. All N- or C-terminus and linker fusion protein variants in which the hCD25/hIL-2Rα moiety was fused to hIL-2 were predicted to exhibit reduced binding of the fusion protein to the CD25 of the hIL-2 receptor on cells. Experimentally these constructs exhibited strongly attenuated hIL-2 activity (by at least 45-fold) on the high affinity TL-2 receptor (NK-92) and by 18-fold on the intermediate hIL-2 receptor (TF1+IL-2Rβ).
1a
1a
0a
0a
0a
a = Fold change is an estimate only since a full four parameter logistic curve was not reached
Since there was no reduction in hIL-2 activity in the various N-terminus or C-terminus immunoglobulin heavy chain fusion proteins, the hIL-2 Cterm heavy chain L6 fusion (SEQ ID NOs: 361, 374), designated as “1H3-hIgG1-L6-hIL-2”, was used as the base construct for antibody-attenuated-hIL-2 fusion protein variants with substitutions in the hIL-2 moiety. Single, double and/or multiple amino acid substitutions were introduced into selected residues of human IL-2 in order to investigate the role those residues play in the recognition of either human CD25/IL-2Rα and/or human CD122/IL-2Rβ or CD132/IL-2Rγ (human IL-2R subunits). Over three hundred antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety were generated and evaluated in 6 rounds. These variants were first screened using a flow-based phosphorylated STAT5 (pSTAT5) assay at a fixed concentration on IL-2 dependent cell lines (NK-92 and TF1+IL-2Rβ) as well as in dose-titration curves. Phosphorylated STAT5 is a downstream signal of IL-2 activity and was used as a snapshot measurement of IL-2 potency. IL-2 dependent cell proliferation assays were also performed to measure IL-2 activity over a period of 3-4 days. Criteria for attenuated hIL-2 selection included: (1) reduced IL-2 potency on both NK-92 and TF1+IL-2Rβ cell lines with greater than 50% agonist activity on both cell lines; and (2) moderate-to-high production yield.
Human anti-DNase I antibody-hIL-2 fusion proteins were generated by fusing the human IL-2 or the human IL-2 variants (SEQ ID NOs: 1-344, 377, 378, and 575) to the C-terminus of a human anti-DNase I antibody (clone 1H3, having a human IgG1 isotype) heavy chain via the L6 linker, which were combined with the hIgG1 light chain (1H3-hkappa LC; SEQ ID NO: 374) to generate the 1H3-hIgG1-L6-hIL-2 fusion proteins (provided in Table 28). Mouse anti-yellow fever virus antibody-hIL-2 fusion proteins were also generated by fusing human IL-2 variants to the C-terminus of a mouse anti-yellow fever virus antibody (clone 2D12, having a mouse IgG1 isotype) heavy chain with a D265A substitution for decreased immune effector function via the L6 linker, which were combined with the 2D12-mIgG1 light chain (2D12-mKappa LC; SEQ ID NO: 376) to generate the 2D12-mIgG1-D265A-L6-hIL-2 fusion proteins (provided in Table 28). Some of these mouse anti-yellow fever virus antibody-hIL-2 fusion proteins were formatted onto a human IgG1 constant region and were generated in the same manner as described above using, which was combined with 2D12-hKappa light chain (2D12-hKappa LC; SEQ ID NO: 573). Iterations of IL-2 amino acid substitutions were performed in six rounds, designated Groups 1 to 6. 1H3-hIgG1-L6-hIL-2, 2D12-mIgG1-D265A-L6-hIL-2, and 2D12-hIgG1-L6-hIL-2 fusion proteins were produced, expressed, and Protein-A purified using standard techniques.
Group 1 contained an initial series of only 2D12-mIgG1-D265A-L6-hIL-2 or 2D12-hIgG1-L6-hIL-2 fusion proteins which comprised a substitution or combination of substitutions in human IL-2 which were predicted to be involved in binding to only one of the IL-2 receptor subunits CD25/IL-2Rα, CD122/IL-2Rβ, or CD132/IL-2Rγ. The fusion proteins in this group included the following substitutions to IL-2 predicted to modulate binding to CD25/IL-2Rα: F42K (SEQ ID NO: 1), V69A (SEQ ID NO: 2), V69E (SEQ ID NO: 3), V69F (SEQ ID NO: 4), V69G (SEQ ID NO: 5), V69H (SEQ ID NO: 6), V69I (SEQ ID NO: 7), V69K (SEQ ID NO: 8), V69L (SEQ ID NO: 9), V69M (SEQ ID NO: 10), V69Q (SEQ ID NO: 11), V69S (SEQ ID NO: 12), V69T (SEQ ID NO: 13), V69W (SEQ ID NO: 14), V69Y (SEQ ID NO: 15), V69R (SEQ ID NO: 581), (F42K/F44K) (SEQ ID NO: 16), (F44K/Y45R) (SEQ ID NO: 17), (F42K/V69R) (SEQ ID NO: 18), (Y45R/V69R) (SEQ ID NO: 19), (F42K/F44K/Y45R) (SEQ ID NO: 20), (F42A/Y45A/L72G) (SEQ ID NO: 574), (R38A/F42K/Y45R) (SEQ ID NO: 21), (R38E/F42K/Y45R) (SEQ ID NO: 22), (K43E/F42K/Y45R) (SEQ ID NO: 23), (K43T/F42K/Y45R) (SEQ ID NO: 24), (F42K/Y45R/E62A) (SEQ ID NO: 25), (P65R/F42K/Y45R) (SEQ ID NO: 26), (P65S/F42K/Y45R) (SEQ ID NO: 27), (V69A/F42K/Y45R) (SEQ ID NO: 28), (V69D/F42K/Y45R) (SEQ ID NO: 29), or (V69R/F42K/Y45R) (SEQ ID NO: 30). The substitutions in this group included the following substitutions predicted to modulate binding to CD122/IL-2Rβ: D20A (SEQ ID NO: 31), D20N (SEQ ID NO: 32), D20K (SEQ ID NO: 33), N88A (SEQ ID NO: 34), N88G (SEQ ID NO: 35), N88H (SEQ ID NO: 36), N88K (SEQ ID NO: 37), (D20A/D84A) (SEQ ID NO: 38), (D20A/E15A) (SEQ ID NO: 39), (D20A/E95A) (SEQ ID NO: 40), (D20A/N88A) (SEQ ID NO: 41), (D20A/S87A) (SEQ ID NO: 42), (D84A/N88A) (SEQ ID NO: 43), (E15A/N88A) (SEQ ID NO: 44), or (S87A/N88A) (SEQ ID NO: 45). Group 1 also included the following substitutions to IL-2 predicted to modulate IL-2 binding to CD132/IL-2-Rγ: Q126L (SEQ ID NO: 377) or Q126E (SEQ ID NO: 378). The IL-2 substitutions studied in Group 1 were not predicted to modulate binding to more than one of the IL-2 receptor subunits.
Group 2 contained a series of 1H3-hIgG1-L6-hIL-2 fusion proteins which comprised one or more substitutions in human IL-2 which were predicted to be involved in CD25/IL-2Rα binding only. The fusion proteins in this group included the following substitutions to IL-2 predicted to modulate binding to CD25/IL-2Rα: R38A (SEQ ID NO: 46), R38D (SEQ ID NO: 47), R38E (SEQ ID NO: 48), R38Q (SEQ ID NO: 49), F42R (SEQ ID NO: 50), F42A (SEQ ID NO: 51), F42D (SEQ ID NO: 52), F42H (SEQ ID NO: 53), K43A (SEQ ID NO: 54), K43E (SEQ ID NO: 55), K43Q (SEQ ID NO: 56), Y45A (SEQ ID NO: 57), Y45K (SEQ ID NO: 58), Y45S (SEQ ID NO: 59), Y45R (SEQ ID NO: 60), E61A (SEQ ID NO: 61), E61R (SEQ ID NO: 62), E61K (SEQ ID NO: 63), E62A (SEQ ID NO: 64), E62R (SEQ ID NO: 65), E62K (SEQ ID NO: 66), E62Y (SEQ ID NO: 67), E68Y (SEQ ID NO: 68), E68A (SEQ ID NO: 69), E68K (SEQ ID NO: 70), E68R (SEQ ID NO: 71), E68L (SEQ ID NO: 72), L72Y (SEQ ID NO: 73), L72R (SEQ ID NO: 74), L72A (SEQ ID NO: 75), L72D (SEQ ID NO: 76), L72H (SEQ ID NO: 77), L72F (SEQ ID NO: 78), (R38D/E61R) (SEQ ID NO: 79), (R38D/E61R/K43E) (SEQ ID NO: 80), or (T3A/F42A/Y45A/L72G/C125A) (SEQ ID NO: 81). The substitution T3A was introduced into the IL-2 amino acid sequence to remove the predicted O-linked glycosylation site on human IL-2 (see for example Int'l Pub. No. WO2012/107417) and the substitution C125A was introduced into the IL-2 amino acid sequence to remove an unpaired cysteine residue (see for example Int'l Pub. No. WO2018/184964). The IL-2 substitutions studied in Group 2 were predicted to not modulate IL-2 binding to CD132/IL-2-Rγ, nor were these substitutions predicted to modulate binding to more than one of the IL-2 receptor subunits.
Group 3 contained a series of 1H3-hIgG1-L6-hIL-2 fusion proteins which comprised one or more substitutions in human IL-2 which were predicted to be involved in CD122/IL-2Rβ binding only. The fusion proteins in this group included the following substitutions to IL-2 predicted to modulate binding to CD122/IL-2Rβ: E15A (SEQ ID NO: 82), E15R (SEQ ID NO: 83), E15K (SEQ ID NO: 84), H16A (SEQ ID NO: 85), H16Y (SEQ ID NO: 86), H16E (SEQ ID NO: 87), L19A (SEQ ID NO: 88), D20I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20T (SEQ ID NO: 92), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), R81A (SEQ ID NO: 97), D84A (SEQ ID NO: 98), D84R (SEQ ID NO: 99), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 107), 192A (SEQ ID NO: 108), 192Y (SEQ ID NO: 109), 192S (SEQ ID NO: 110), I92F (SEQ ID NO: 111), 192R (SEQ ID NO: 112), I92D (SEQ ID NO: 113), I92E (SEQ ID NO: 114), E95A (SEQ ID NO: 115), E95R (SEQ ID NO: 116), E95K (SEQ ID NO: 117), (D20Y/H16E) (SEQ ID NO: 118), (D20Y/H16A) (SEQ ID NO: 119), (D20Y/H16Y) (SEQ ID NO: 120), (D20Y/I92A) (SEQ ID NO: 121), (D20Y/I92S) (SEQ ID NO: 122), (D20Y/I92R) (SEQ ID NO: 123), (D20Y/E95R) (SEQ ID NO: 124), or (D20Y/E95A) (SEQ ID NO: 125).
Group 4 contained a series of fusion proteins containing the 1H3-hIgG1-L6-hIL-2 HC fused to a CD25/IL-2Rα extracellular domain moiety (SEQ ID NO: 126), a 20 amino acid linker (L20) (SEQ ID NO: 364), and human IL-2 variants comprising one or more substitutions to residues predicted to be involved in binding to CD122/IL-2Rβ. The fusion proteins in this group included the following substitutions to IL-2 predicted to modulate binding to CD122/IL-2Rβ: E15A (SEQ ID NO: 82), D20I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 107), I92A (SEQ ID NO: 108), E95A (SEQ ID NO: 115), or E95K (SEQ ID NO: 117). The antibody-attenuated hIL-2 fusion proteins in this group are denoted as 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL-2.
Group 5 contained a series of 1H3-hIgG1-L6-hIL-2 which comprised a combination of substitutions in IL-2 which were predicted to be involved in binding of IL-2 to CD25/IL-2Rα and to CD122/IL-2Rβ or CD132/IL-2Rγ. In addition, some variants had a deletion in the first three amino acids at the N-terminus of the hIL-2 moiety (Δ1-3APT). The fusion proteins in Group 5 included the following substitutions to IL-2 predicted to modulate IL-2 binding to CD25/IL-2Rα and to CD122/IL-2Rβ: (F42D/D20A) (SEQ ID NO: 127), (F42R/D20A) (SEQ ID NO: 128), (F42K/D20A) (SEQ ID NO: 129), (F42A/D20A) (SEQ ID NO: 130), (F42H/D20A) (SEQ ID NO: 131), (Y45R/D20A) (SEQ ID NO: 132), (Y45K/D20A) (SEQ ID NO: 133), (R38N/D20A) (SEQ ID NO: 134), (R38G/D20A) (SEQ ID NO: 135), (R38H/D20A) (SEQ ID NO: 136), (R381/D20A) (SEQ ID NO: 137), (R38L/D20A) (SEQ ID NO: 138), (R38M/D20A) (SEQ ID NO: 139), (R38F/D20A) (SEQ ID NO: 140), (R38P/D20A) (SEQ ID NO: 141), (R38S/D20A) (SEQ ID NO: 142), (R38T/D20A) (SEQ ID NO: 143), (R38W/D20A) (SEQ ID NO: 144), (R38Y/D20A) (SEQ ID NO: 145), (R38V/D20A) (SEQ ID NO: 146), (R38A/D20A) (SEQ ID NO: 147), (R38Q/D20A) (SEQ ID NO: 148), (D20A/R38E) (SEQ ID NO: 149), (R38D/D20A) (SEQ ID NO: 150), (K43E/D20A) (SEQ ID NO: 151), (E61A/D20A) (SEQ ID NO: 152), (E62A/D20A) (SEQ ID NO: 153), (E62Y/D20A) (SEQ ID NO: 154), (L72D/D20A) (SEQ ID NO: 155), (L72H/D20A) (SEQ ID NO: 156), (L72R/D20A) (SEQ ID NO: 157), (F42D/I92D) (SEQ ID NO: 158), (F42R/I92D) (SEQ ID NO: 159), (F42H/I92D) (SEQ ID NO: 160), (F42A/I92D) (SEQ ID NO: 161), (H16A/F42A) (SEQ ID NO: 575), (K43E/I92D) (SEQ ID NO: 162), (Y45R/I92D) (SEQ ID NO: 163), (Y45K/I92D) (SEQ ID NO: 164), (E62A/I92D) (SEQ ID NO: 165), (E62Y/I92D) (SEQ ID NO: 166), (L72D/I92D) (SEQ ID NO: 167), (L72H/I92D) (SEQ ID NO: 168), (L72R/I92D) (SEQ ID NO: 169), (R38D/I92D) (SEQ ID NO: 170), (R38E/I92D) (SEQ ID NO: 171), (R38Q/I92D) (SEQ ID NO: 172), (R38A/I92D) (SEQ ID NO: 173), (R38E/N88R) (SEQ ID NO: 174), (R38E/D84R) (SEQ ID NO: 175), (R38E/D84K) (SEQ ID NO: 176), (F42A/Y45R/D20A) (SEQ ID NO: 177), (F42H/Y45R/D20A) (SEQ ID NO: 178), (R38D/E61R/D20A) (SEQ ID NO: 179), (R38E/E61R/D20A) (SEQ ID NO: 180), (R38Q/E61R/D20A) (SEQ ID NO: 181), (R38A/E61R/D20A) (SEQ ID NO: 182), (R38A/D20A/E95A) (SEQ ID NO: 183), (D20A/E95A/R38D) (SEQ ID NO: 184), (D20A/E95A/R38E) (SEQ ID NO: 185), (D20A/E95A/R38Q) (SEQ ID NO: 186), (D20A/E95A/F42R) (SEQ ID NO: 187), (D20A/E95A/F42A) (SEQ ID NO: 188), (D20A/E95A/F42D) (SEQ ID NO: 189), (D20A/E95A/F42H) (SEQ ID NO: 190), (D20A/E95A/F42K) (SEQ ID NO: 191), (D20A/E95A/K43A) (SEQ ID NO: 192), (D20A/E95A/K43E) (SEQ ID NO: 193), (D20A/E95A/K43Q) (SEQ ID NO: 194), (D20A/E95A/Y45A) (SEQ ID NO: 195), (D20A/E95A/Y45K) (SEQ ID NO: 196), (D20A/E95A/Y45S) (SEQ ID NO: 197), (D20A/E95A/Y45R) (SEQ ID NO: 198), (D20A/E95A/E61A) (SEQ ID NO: 199), (D20A/E95A/E62A) (SEQ ID NO: 200), (D20A/E95A/E62R) (SEQ ID NO: 201), (D20A/E95A/E62K) (SEQ ID NO: 202), (D20A/E95A/E62Y) (SEQ ID NO: 203), (D20A/E95A/E68Y) (SEQ ID NO: 204), (D20A/E95A/E68A) (SEQ ID NO: 205), (D20A/E95A/E68L) (SEQ ID NO: 206), (D20A/E95A/L72Y) (SEQ ID NO: 207), (D20A/E95A/L72R) (SEQ ID NO: 208), (D20A/E95A/L72A) (SEQ ID NO: 209), (D20A/E95A/L72D) (SEQ ID NO: 210), (D20A/E95A/L72H) (SEQ ID NO: 211), (D20A/E95A/L72F) (SEQ ID NO: 212), (F42K/Y45R/D20A/S87A) (SEQ ID NO: 213), (F42K/Y45R/D20A/E95A) (SEQ ID NO: 214), (D20A/R38E/C125A) (SEQ ID NO: 215), (T3A/D20A/R38E) (SEQ ID NO: 216), (T3A/D20A/R38E/C125A) (SEQ ID NO: 217), (Δ1-3APT/D20A/R38E) (SEQ ID NO: 218), or (Δ1-3APT/D20A/R38E/C125A) (SEQ ID NO: 219). The fusion proteins in Group 5 included the following substitutions to IL-2 predicted to modulate IL-2 binding to CD25/IL-2Rα and to CD132/IL-2R: (R38E/Q22A) (SEQ ID NO: 220), (R38E/T123A) (SEQ ID NO: 221), (R38E/I129A) (SEQ ID NO: 222), (R38E/S130A) (SEQ ID NO: 223), (R38E/Q126A) (SEQ ID NO: 224), (R38E/Q126D) (SEQ ID NO: 225), (R38E/Q126V) (SEQ ID NO: 226), (R38E/Q22A/S130A) (SEQ ID NO: 227), (F42K/Y45R/Q126D) (SEQ ID NO: 228), or (D20A/E95A/Q126D) (SEQ ID NO: 229). Mutations to the hIL-2 sequence for Group 5 antibody-attenuated hIL-2 fusion proteins in which the numbering is according to IL-2 sequence are listed in SEQ ID NO: 127-229 and 575.
Group 6 contained a series of 1H3-hIgG1-L6-hIL-2 fusion proteins which comprised a combination of substitutions in human IL-2 which were predicted to be involved in binding of IL-2 to CD25/IL-2Rα and to CD122/IL-2Rβ, but not to CD132/IL-2Rγ. The fusion proteins in Group 6 included the following combination of substitutions in IL-2 predicted to modulate IL-2 binding to CD25/IL-2Rα and CD122/IL-2Rβ: (D20A/E61R) (SEQ ID NO: 230), (D20A/E61N) (SEQ ID NO: 231), (D20A/E61D) (SEQ ID NO: 232), (D20A/E61Q) (SEQ ID NO: 233), (D20A/E61G) (SEQ ID NO: 234), (D20A/E61H) (SEQ ID NO: 235), (D20A/E61I) (SEQ ID NO: 236), (D20A/E61L) (SEQ ID NO: 237), (D20A/E61K) (SEQ ID NO: 238), (D20A/E61M) (SEQ ID NO: 239), (D20A/E61F) (SEQ ID NO: 240), (D20A/E61P) (SEQ ID NO: 241), (D20A/E61S) (SEQ ID NO: 242), (D20A/E61T) (SEQ ID NO: 243), (D20A/E61W) (SEQ ID NO: 244), (D20A/E61Y) (SEQ ID NO: 245), (D20A/E61V) (SEQ ID NO: 246), (D20A/F42N) (SEQ ID NO: 247), (D20A/F42Q) (SEQ ID NO: 248), (D20A/F42E) (SEQ ID NO: 249), (D20A F42G) (SEQ ID NO: 250), (D20A/F42I) (SEQ ID NO: 251), (D20A/F42L) (SEQ ID NO: 252), (D20A/F42M) (SEQ ID NO: 253), (D20A/F42P) (SEQ ID NO: 254), (D20A/F42S) (SEQ ID NO: 255), (D20A/F42T) (SEQ ID NO: 256), (D20A/F42W) (SEQ ID NO: 257), (D20A/F42Y) (SEQ ID NO: 258), (D20A/F42V) (SEQ ID NO: 259), (D20A/Y45A) (SEQ ID NO: 260), (D20A/Y45N) (SEQ ID NO: 261), (D20A/Y45D) (SEQ ID NO: 262), (D20A/Y45Q) (SEQ ID NO: 263), (D20A/Y45E) (SEQ ID NO: 264), (D20A/Y45G) (SEQ ID NO: 265), (D20A/Y45H) (SEQ ID NO: 266), (D20A/Y45I) (SEQ ID NO: 267), (D20A/Y45L) (SEQ ID NO: 268), (D20A/Y45M) (SEQ ID NO: 269), (D20A/Y45F) (SEQ ID NO: 270), (D20A/Y45P) (SEQ ID NO: 271), (D20A/Y45S) (SEQ ID NO: 272), (D20A/Y45T) (SEQ ID NO: 273), (D20A/Y45W) (SEQ ID NO: 274), (D20A/Y45V) (SEQ ID NO: 275), (I92D/F42N) (SEQ ID NO: 276), (I92D/F42Q) (SEQ ID NO: 277), (I92D/F42E) (SEQ ID NO: 278), (I92D/F42G) (SEQ ID NO: 279), (I92D/F42I) (SEQ ID NO: 280), (I92D/F42L) (SEQ ID NO: 281), (I92D/F42K) (SEQ ID NO: 282), (I92D/F42M) (SEQ ID NO: 283), (I92D/F42P) (SEQ ID NO: 284), (I92D/F42S) (SEQ ID NO: 285), (I92D/F42T) (SEQ ID NO: 286), (I92D/F42W) (SEQ ID NO: 287), (I92D/F42Y) (SEQ ID NO: 288), (I92D/F42V) (SEQ ID NO: 289), (I92D/Y45A) (SEQ ID NO: 290), (I92D/Y45N) (SEQ ID NO: 291), (I92D/Y45D) (SEQ ID NO: 292), (I92D/Y45Q) (SEQ ID NO: 293), (I92D/Y45E) (SEQ ID NO: 294), (I92D/Y45G) (SEQ ID NO: 295), (I92D/Y45H) (SEQ ID NO: 296), (I92D/Y45I) (SEQ ID NO: 297), (I92D/Y45L) (SEQ ID NO: 298), (I92D/Y45M) (SEQ ID NO: 299), (I92D/Y45F) (SEQ ID NO: 300), (I92D/Y45P) (SEQ ID NO: 301), (I92D/Y45S) (SEQ ID NO: 302), (I92D/Y45T) (SEQ ID NO: 303), (I92D/Y45W) (SEQ ID NO: 304), (I92D/Y45V) (SEQ ID NO: 305), (R38E/D20H) (SEQ ID NO: 306), (R38E/D20S) (SEQ ID NO: 307), (F42A/N88R) (SEQ ID NO: 308), (F42A/N88D) (SEQ ID NO: 309), (R38E/D84A) (SEQ ID NO: 310), (R38E/D84N) (SEQ ID NO: 311), (R38E/D84Q) (SEQ ID NO: 312), (R38E/D84E) (SEQ ID NO: 313), (R38E/D84G) (SEQ ID NO: 314), (R38E/D84H) (SEQ ID NO: 315), (R38E/D84I) (SEQ ID NO: 316), (R38E/D84L) (SEQ ID NO: 317), (R38E/D84M) (SEQ ID NO: 318), (R38E/D84F) (SEQ ID NO: 319), (R38E/D84P) (SEQ ID NO: 320), (R38E/D84S) (SEQ ID NO: 321), (R38E/D84T) (SEQ ID NO: 322), (R38E/D84W) (SEQ ID NO: 323), (R38E/D84Y) (SEQ ID NO: 324), (R38E/D84V) (SEQ ID NO: 325), (R38E/I92A) (SEQ ID NO: 326), (R38E/I92R) (SEQ ID NO: 327), (R38E/I92N) (SEQ ID NO: 328), (R38E/I92Q) (SEQ ID NO: 329), (R38E/I92E) (SEQ ID NO: 330), (R38E/I92G) (SEQ ID NO: 331), (R38E/I92H) (SEQ ID NO: 332), (R38E/I92L) (SEQ ID NO: 333), (R38E/I92K) (SEQ ID NO: 334), (R38E/I92M) (SEQ ID NO: 335), (R38E/I92F) (SEQ ID NO: 336), (R38E/I92P) (SEQ ID NO: 337), (R38E/I92S) (SEQ ID NO: 338), (R38E/192T) (SEQ ID NO: 339), (R38E/I92W) (SEQ ID NO: 340), (R38E/I92Y) (SEQ ID NO: 341), (R38E/192V) (SEQ ID NO: 342), (R38E/H16E) (SEQ ID NO: 343), or (R38K/D20A) (SEQ ID NO: 344). Mutations to the hIL-2 sequence for Group 6 antibody-attenuated hIL-2 fusion proteins in which the numbering is according to IL-2 sequence is listed in SEQ ID NO: 230-344.
The binding kinetics of some purified 1H3-hIgG1-L6-hIL-2 variant proteins for individual recombinant human CD25 and human CD122 were determined using bio-layer interferometry (BLI). Briefly, binding experiments were performed using an Octet Red96 instrument (Pall Forteio) at 25° C. C-terminal poly-histidine tagged human CD25 and human CD122 extracellular domains were captured onto anti-His2 sensors (Pall Forteio). Receptor loaded sensors were dipped into a 7-point serial 3-fold dilution of each 1H3-hIgG-L6-hIL-2 variant, starting at a top concentration of 300 nM. 1H3-hIgG2-L6-hIL-2 fusion proteins were diluted into an assay buffer consisting of phosphate buffered saline (PBMS) supplemented with 0.100 BSA, 0.02% Tween-20 (pH 7.2). Loaded sensors were regenerated using 10 mM Glycine buffer (pH 1.7). Kinetic constants were calculated using a monovalent binding model.
Table 2 documents the association constant (kon), dissociation constant (koff), and equilibrium constant (KD) of 74 immunoglobulin-hIL-2 fusion protein variants bound to recombinant human CD25 or recombinant human CD122.
Table 3 documents the association (kon) constants, dissociation (koff) constants, and equilibrium constants (KD) of 74 1H3-hIgG1-L6-hIL-2 fusion proteins bound to recombinant human CD122.
aSEQ ID NO: 345 corresponds to wild type hIL-2.
bSEQ ID NO: 57 is attenuated IL-2 sequence only.
The attenuation of antibody-attenuated hIL-2 fusion proteins described in Example 2 was tested in a fixed concentration pSTAT5 screen using the NK-92 (expressing the high affinity hIL-2 receptor) and TF1+IL-2Rβ (expressing the intermediate affinity hIL-2 receptor) cell lines as described in Protocol D. Tables 4-8 list the fold change of geometric mean fluorescent intensity (gMFI) of antibody-attenuated hIL-2 fusion proteins from free cytokine wild-type rhIL-2, a measurement of reduction of IL-2 activity. For the fixed concentration screen, the fold change was calculated by dividing the gMFI of the rhIL-2 by the gMFI of the variants. For experiments with full titration curves, fold change from rhIL-2 was calculated by dividing the EC50 values for the rhIL-2 by the EC50 of variants. Fold change was rounded to the nearest whole number. A reduced gMFI in both NK-92 and TF1+IL-2Rβ cell lines when compared to the gMFI resulting from rhIL-2 was indicative of attenuation of IL-2 activity at both the high- and intermediate-affinity receptors. Group 1 variants described in Example 2 were not tested in the fixed concentration cell-based potency pSTAT5 screen.
Each variant tested was also assessed for IL-2 agonistic activity and characterized either as a full or partial IL-2 agonist, or having no IL-2 activity (inactive). 1H3-hIgG1-L6-hIL-2 fusion protein dose-titration curves that reached the maximal gMFI level exhibited by the rhIL-2 positive control were considered to be antibody-attenuated hIL-2 fusion protein with full agonist activity. Partial agonist activity was calculated as a percentage of full activity using rhIL-2 maximal gMFI as 100%. Antibody-attenuated hIL-2 fusion protein with less than 10% of the rhIL-2 maximal gMFI at the highest concentration of 1200 nM were considered to have no agonist activity (inactive). Some EC50 values and level of attenuation could not be accurately calculated using the GraphPad Prism 7 software since activity did not reach a maximum and accordingly these values are an estimate.
pSTAT5 fixed concentration results demonstrated that while some single residue substitutions attenuated IL-2 activity on the high-affinity cell line (NK-92), a combination of substitutions which modulated binding to both the alpha chain and the beta chain or both the alpha chain and gamma chain were required to substantially attenuate IL-2 activity on the high affinity IL-2 receptor (more than 20-fold attenuation from recombinant hIL-2).
The attenuation of selected antibody-attenuated hIL-2 fusion proteins described in Example 2 (1H3-hIgG1-L6-hIL-2 fusion protein from Groups 2-6) were tested in pSTAT5 titration curves using the NK-92 and TF1+IL-2Rβ cell lines as described in Protocol D.
The gMFI of the Alexa Fluor 647 pSTAT5-positive signal was used to generate four parameter logistic curves and GraphPad Prism 7 software was then used to calculate EC50 values. These values were compared to recombinant hIL-2 (rhIL-2) control as a measurement of attenuation. Tables 9-13 summarize the fold change in activity from rhIL-2 calculated using the gMFI of the Alexa Fluor 647 signal.
An increase in the fold change from rhIL-2 was indicative of the degree of attenuation of hIL-2 activity. Each antibody-attenuated hIL-2 fusion protein tested in the pSTAT5 titration curve was also assessed for agonistic activity and characterized as either full, partial, or no activity (inactive). Antibody-attenuated hIL-2 fusion protein dose-titration curves that reached the maximal gMFI level as the rhIL-2 were considered to be variants with full agonist activity. Partial agonist activity was calculated as described in Example 3. Inactive antibody-attenuated hIL-2 fusion proteins were classified as having less than 10% activity in comparison to rhIL-2. Some fold changes from rhIL-2 could not be accurately calculated (denoted as Not Calculated or “NC”) using the GraphPad Prism 7 software since a full four parameter logistic curve was not generated and accordingly these values are an estimate (annotated as a in Tables 9-13). However, these variants had greater than 10,000-fold attenuation from rhIL-2 on graphs (data not shown). This is denoted on Tables 9-13 as “>10,000 on graph; NC”.
Full titration pSTAT5 curves demonstrated similar findings as presented in Example 3 in which substitutions that modulated binding to both the alpha chain and the beta chain substantially attenuated IL-2 activity on the high affinity IL-2 receptor in comparison to single substitutions for binding to the alpha or beta chain only. The full titration pSTAT5 assay was additionally able to differentiate between variants with substitutions that caused inactivity versus highly attenuated variants. Finally, comparison of dose-titration curves illustrated more accurate of levels of attenuation over a fixed concentration assay.
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
The attenuation of IL-2 activity of antibody-attenuated hIL-2 fusion proteins from Groups 1-6 in Example 2 (2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, and 1H3-hIgG1-L6-hIL-2 fusion proteins) were tested in proliferation assays in both the NK-92 and TF1+IL-2Rβ cell lines as described in Protocol E. The results of the assays are provided in Tables 14-19.
Selected 1H3-hIgG1-L6-hIL-2 fusion proteins with substantial attenuation in the pSTAT5 titration curves from Example 4 were tested in this cell-based proliferation assay. pSTAT5 is a downstream read-out of IL-2 activity and assays require only 10 minutes of stimulation which may be a small snapshot of IL-2 dependent activity. For proliferation assays, cells were incubated with 2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, 1H3-hIgG1-L6-hIL-2 fusion proteins, or recombinant hIL-2 control for 3-4 days, providing a more physiological relevant read-out of IL-2 dependent activity in vivo. Other 2D12-mIgG1-D265A-L6-hIL-2 and 2D12-hIgG1-L6-hIL-2 fusion proteins that were generated but not tested in a pSTAT5 assay were assayed for IL-2 dependent activity using this proliferation assay.
Similar to cell-based pSTAT5 dose-titration experiments, the calculated EC50 as determined from relative luminescence units (RLU) instead of gMFI and analysis of the results were performed identically to Example 4 once EC50 was calculated. Similar to results identified in Example 4, proliferation curves demonstrated that some substitutions that modulated binding to both the alpha chain and beta chain substantially attenuated IL-2 activity on the high affinity receptor in comparison to single substitutions for binding to the alpha or beta chain only. These selected 1H3-hIgG1-L6-hIL-2 fusion proteins were also tested for proliferation on the TF1+IL-2Rβ cell line and demonstrated that some of these same substitutions substantially attenuated IL-2 activity on the intermediate affinity receptor.
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
a Fold change is an estimate only since a full four parameter logistic curve was not reached
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a Fold change is an estimate only since a full four parameter logistic curve was not reached
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a Fold change is an estimate only since a full four parameter logistic curve was not reached
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a Fold change is an estimate only since a full four parameter logistic curve was not reached
Several approaches were used to generate a variety of different anti-hPD-1 antibodies with desired properties.
In one approach, anti-hPD-1 human monoclonal antibodies were generated using transgenic chickens (OmniChicken™) that express human antibody genes (human light chain (VLCL or VKCK) and human VH) and the chicken constant regions of the heavy chain (Ching et al., mAbs 2018). Transgenic chickens were immunized with 100 μg of Fc-tagged human PD-1 protein (huPD-1-Fc) (SEQ ID NO: 380) every 14 days for 14 weeks. In another approach, transgenic chickens were genetically immunized six times with DNA encoding human PD-1 (SEQ ID NO: 347) followed by a final boost with 100 μg huPD-1-Fc (SEQ ID NO: 380). The serum immune response of each animal was monitored by ELISA against biotinylated human PD-1 on streptavidin coated plates.
Splenocytes were isolated from each immunized animal, tested for positive antibody clones using the Gel Encapsulated Microenvironment (GEM) assay (as described in Mettler Izquierdo, S., Varela, S., Park, M., Collarini, E. J., Lu, D., Pramanick, S., Rucker, J., Lopalco, L., Etches, R., & Harriman, W. (2016). High-efficiency antibody discovery achieved with multiplexed microscopy. Microscopy (Oxford, England), 65(4), 341-352) and screened against human PD-1 labelled beads. Positive clones were sequenced and variable regions of the heavy and light chains were cloned, assembled into a single chain variable fragment, and fused to the hinge and Fc regions of immunoglobulins (ScFv-Fc). These unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells and supernatants were tested for binding activity by ELISA on plates coated with huPD-1-Fc (SEQ ID NO: 380) or cynomolgous-PD-1-Fc (SEQ ID NO: 381). In total, 102 unique anti-human PD-1 variable heavy and variable light pairings were identified using this method. 2H7-hIgG4 (SEQ ID NOs: 382-391, 424, and 425) and A2-hIgG4 (SEQ ID NOs: 402-411, 428, and 429) were among the antibodies identified in this approach.
Other approaches led to the identification of an anti-hPD-1 antibody denoted as C51E6-hIgG4, which was germline optimized to become the antibody designated C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426, 427), and humanized and further sequence optimized to become the antibody designated Abz1mod-hIgG4 (SEQ ID NOs: 449, 450).
The anti-PD-1 variable region sequences were expressed as human IgG4 kappa antibodies and were evaluated for the ability to bind to PD-1 expressing cells using flow cytometry as described in General Methods Protocol A. Antibodies to be tested were first screened for binding to human PD-1 using a Jurkat cell line expressing recombinant human PD-1 (Jurkat+hPD-1 cell line). Antibodies were serially diluted from a top concentration of 280 nM and Allophycocyanin-conjugated anti-human IgG secondary antibody was then added to cells for detection. Of 92 hits, 79 test anti-PD-1 antibodies had an EC50 binding (by flow cytometry) of <30 nM. 2H7-hIgG4 (SEQ ID NOs: 382-391, 424, and 425), C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426, and 427), A2-hIgG4 (SEQ ID NOs: 402-411, 428, and 429), OMC.1.B6-hIgG4 (SEQ ID NOs: 438 and 439), OMC.1.D6-hIgG4 (SEQ ID NOs: 442 and 443), OMC.2.C6-hIgG4 (SEQ ID NOs: 440 and 441), 1H9-hIgG4 (SEQ ID NOs: 576 and 525), 1D5-hIgG4 (SEQ ID NOs: 577 and 527), and 2A3.H7-hIgG4 (SEQ ID NOs: 424 and 523) were among a group of antibodies identified as antibodies with medium to high affinity binding to hPD-1 using a Jurkat cell line expressing human PD-1 (SEQ ID NO: 346). The calculated EC50 of binding to Jurkat cells which recombinantly expressed hPD-1 by flow cytometry in multiple experiments was 0.1-0.3 nM for 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4. The calculated EC50 of binding to Jurkat cells expressing hPD-1 by flow for C51E6-5-hIgG4 was 2-4 nM, and 3-16 nM for A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4. Binding was specific to hPD-1 since 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, 1H9-hIgG4, 1D5-hIgG4, 2A3.H7-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4 antibody titrations did not bind the parental Jurkat cell line which did not express hPD-1 (data not shown).
2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 were assessed for binding competition to hPD-1 in the presence of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A as described in General Methods Protocol B.
As a control, OPDIVO® (nivolumab) was titrated in the presence of saturating concentrations of 10 μM anti-hPD-1 #1-mIgG2b-N297A (
Anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 were tested for PD-1 antagonist activity using an in vitro cell-based human PD-1/PD-L1 blockade bioassay as described in General Methods Protocol C. All antibodies except A2-hIgG4 were tested at 200 nM final concentration. A2-hIgG4 was tested at 500 nM final concentration.
None of the anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, OMC.2.C6-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4 demonstrated hPD-1 antagonist activity, as all displayed luminescence levels of an average of 3000 relative luminescence units (RLU) and exhibited an RLU similar to the negative control KLH-C3-hIgG4 (data not shown). In contrast, the anti-hPD-1 #1, which is a known hPD-1 antagonist that blocks hPD-L1 (SEQ ID NO: 584) engagement with hPD-1, exhibited luminescence of above 14,000 RLU (data not shown).
In order to construct various antibody and antibody-attenuated hIL-2 fusion protein expression vectors, the corresponding polynucleotide encoding sequences of antibody, cytokines, cytokine receptors and linkers were generated and cloned into expression vectors. The antibodies or antibody fusion proteins were transiently expressed in Human Embryonic Kidney (HEK) 293 cells, then purified by affinity chromatography using Protein A- or Protein G-Sepharose. The purified proteins were concentrated and buffer-exchanged to phosphate buffered saline or phosphate buffered saline containing 100 mM L-arginine and 10 mM L-histidine using ultracentrifugal filtration, after which protein concentration was determined.
In some approaches, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 carrying an S228P hinge stabilization mutation were directly fused (df) to hIL-2 or fused to hIL-2 at the C-terminus of the immunoglobulin heavy chain using the L6 linker. An illustration of these anti-PD-1-attenuated hIL-2 fusion proteins is summarized in
The generation of anti-hPD-1-attenuated hIL-2 fusion proteins did not reduce binding to hPD-1, and the anti-hPD-1-attenuated hIL-2 fusion proteins were still able to bind to Jurkat cells expressing human PD-1. The calculated EC50 of tested anti-hPD-1-attenuated hIL-2 fusion proteins in comparison to respective anti-hPD-1 antibody without the attenuated hIL-2 moiety is summarized in Table 20.
The addition of the attenuated hIL-2 moiety on anti-hPD-1 antibodies did not abrogate binding to human PD-1 as demonstrated by a less than 2-fold increase in EC50 binding of anti-hPD-1-hIL-2 fusion proteins to Jurkat+hPD-1 cells in comparison to the anti-hPD-1 antibody without the attenuated hIL-2 moiety.
Anti-hPD-1-attenuated hIL-2 fusion proteins were tested for binding to the hPD-1 receptor in the presence of anti-hPD-1 #1 and anti-hPD-1 #2 as described in General Methods Protocol B and Example 7. The converse experiment was also performed in which anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A was examined for binding to hPD-1 in the presence of saturating concentrations of test antibody-attenuated hIL-2 fusion proteins. In this format, Jurkat cells expressing hPD-1 were plated at 100,000 cells per well in FACS buffer, blocked with anti-human FcγR Blocking Reagent (Miltenyi) for 10 minutes at 4° C. and washed. Test antibody-attenuated hIL-2 fusion proteins 2H7-hIgG4-df-hIL-2 (D20A/R38E), C51E6-5-hIgG4-L6-hIL-2 (D20A/R38E), A2-hIgG4-df-hIL-2 (D20A/R38E), H7-767, and isotype control anti-DNase 1H3-hIgG4-df-hIL-2 (D20A/R38E) were diluted to 280 nM final concentration in 100 μL FACS buffer and incubated with Jurkat cells expressing hPD-1 cells for 1 hour on ice. Cells were washed and re-suspended in FACS buffer containing six-fold serial titrations of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A starting at a maximum concentration of 50 nM for 1 hour on ice. Cells were washed and re-suspended in 1:100 dilution of Phycoerythrin-conjugated anti-mouse IgG light chain kappa monoclonal antibody for 45 minutes on ice. Cells were again washed and re-suspended in FACS buffer with 1:1000 dilution of Sytox Green (Thermo Fisher). Flow cytometry analysis was performed using the BD FACS Canto II (BD Biosciences) and gMFI calculated using FlowJo software version 10. EC50 values were calculated from the gMFI of the Phycoerythrin signal across the titrated concentrations using GraphPad Prism 7 software.
The addition of the attenuated hIL-2 to anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not diminish the ability of the anti-hPD-1 proteins to bind to human PD-1 in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A, similar to the results described in Example 7 (
Anti-hPD-1-attenuated hIL-2 fusion proteins were tested in flow cytometry for binding to cynomolgus PD-1 using a human Embryonic Kidney 293 cell line expressing the SV40 large T cell antigen (HEK-293T) that was transiently transfected to recombinantly express cynomolgus PD-1. For each transfection reaction, 2 million HEK-293T cells were transfected with 2 μg of pCMV6-hygro-HA-cyno-PD-1 (1-185) (SEQ ID NO: 448), a mammalian vector comprising the cynomolgus PD-1 extracellular domain tagged with a human influenza hemagglutinin and the sequence encoding for hygromycin resistance. Transfection was performed by electroporation. Transfected cells were blocked with human FcγR blocking reagent and stained with titrating amounts of anti-hPD-1-attenuated hIL-2 fusion proteins. Additionally, Phycoerythrin conjugated anti-hemagglutinin clone 15B12 was added to cells to stain for transfected cells and Allophycocyanin-conjugated anti-human IgG Fc secondary clone HP6017 (BioLegend Cat #409306) was added to cells to stain bound antibody. The cells were analyzed on the BD Canto II and FlowJo software version 10 was used to gate on live, transfected (hemagglutinin-positive) cells and to calculate gMFI of the Allophycocyanin signal. EC50 values were calculated from the gMFI across the titrated concentrations using GraphPad Prism 7 software.
Anti-hPD-1-attenuated hIL-2 fusion proteins bound to cynomolgus PD-1-expressing HEK-293T cells in a similar fashion to the binding profile seen on Jurkat T cells expressing human PD-1 (
The binding of anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins on activated primary T cells expressing hPD-1 was examined by flow cytometry. To test if 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 bound to native hPD-1, cryopreserved human peripheral blood mononuclear cells (PBMCs) were thawed and activated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin to up-regulate the hPD-1 receptor. Activated PBMCs were collected, blocked with 1:50 dilution of Human FcγR Blocking Reagent (Miltenyi) for 10 minutes at 4° C., and stained with titrated concentrations of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD-1 #1, and isotype control. Cells were then stained with 1:20 dilution of Allophycocyanin-conjugated anti-human IgG Fc to detect bound antibody. To delineate immune subsets, a cocktail of surface markers included anti-human CD3, anti-CD4, and anti-CD8 antibodies was used. In addition, a sample fraction was examined for cellular expression of hPD-1, hCD25, hCD122, and hCD132. Cells were analyzed on the BD Fortessa (BD Biosciences), FlowJo software version 10 was used to gate on T cell subsets then calculate gMFI of the allophycocyanin signal. EC50 values were calculated from the gMFI across the titrated concentrations using GraphPad Prism 7 software. To test the binding of anti-hPD-1-hIL-2 fusion proteins, cryopreserved CD3+ T cells were activated with PMA/ionomycin and flow cytometry binding was performed identically as described above.
Human PD-1 antibody-attenuated hIL-2 fusion proteins were also tested for binding to activated cynomolgus T cells using flow cytometry. Cynomolgus PBMCs were activated with a mixture of 0.081 μM PMA and 1.34 μM ionomycin. 24 hours later, cells were stained using the same procedure as binding to human PD-1 primary cells described above except cynomolgus cross-reactive markers were used. FlowJo software version 10 was used to gate on live, CD3+CD4+ or CD3+CD8+ T cells and then to calculate gMFI of the Allophycocyanin signal. EC50 values were calculated from the gMFI across the titrated concentrations of anti-hPD-1 antibodies or hPD-1 antibody-attenuated hIL-2 fusion proteins using GraphPad Prism 7 software.
In some variants tested, the attenuated hIL-2 also included the substitutions T3A and C125A, which remove a site for O-linked glycosylation and substitute away a free cysteine residue, respectively.
40-50% of CD4+ T cells were PD-1+ while 30-40% of CD8+ T cells were PD-1+ after PMA and ionomycin activation (data not shown). The calculated EC50 for binding to activated human CD3+CD4+ T cells by flow cytometry was 0.1-0.7 nM for 2H7-hIgG4, 12 nM for C51E6-5-hIgG4, 30 nM for A2-hIgG4, and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). The EC50 for binding to activated human CD3+CD8+ T cells was 0.1-0.8 nM for 2H7-hIgG4, 16 nM for C51E6-5-hIgG4, 22 nM for A2-hIgG4, and 0.03 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). The EC50 for binding to activated human CD3+CD4+ T cells was 0.19 nM and activated human CD3+CD8+ T cells was 0.12 nM for H7-767. The EC50 for binding to activated cynomolgus CD3+CD4+ T cells was 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). EC50 for binding to activated cynomolgus CD3+CD8+ T cells was 0.08 nM for 2H7-hIgG4 and 0.03 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). The EC50 for binding to activated cynomolgus CD3+CD4+ T cells was 0.26 nM and activated cynomolgus CD3+CD8+ T cells was 0.24 nM for H7-767. This data demonstrated that when the hPD-1 antibodies were converted to anti-hPD-1-attenuated hIL-2 fusion proteins, the calculated EC50 value for binding to activated hPD-1 remained similar to the calculated EC50 value of hPD-1 naked antibody binding to hPD-1.
H7-767 and H7-632-hIgG1-LAGA anti-PD-1 naked antibody were tested for binding on primary non-activated human CD4+ and CD8+ T cells by flow cytometry. Frozen human CD3+ T were thawed and flow cytometry performed as described above. Both H7-767 III and H7-632-hIgG1-LAGA anti-PD-1 naked antibody did not bind non-activated human CD4+ and CD8+ T cells (data not shown).
Surface plasmon resonance binding analysis was performed using a high-throughput SPR Carterra® LSA™ to determine binding affinities of anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins. Proteins were diluted to 2 or 10 μg/mL in 10 mM sodium acetate pH 4.5 containing 0.01% Tween-20 and coupled to a HC30M (Carterra Bio) chip using sulpho-N-hydroxysuccinimide/1-ethyl-3-(3-dimethylamino) propyl carbodiimide (sulpho-NHS/EDC) coupling chemistry and blocked with ethanolamine. A non-regenerative kinetic coupling process was used to determine binding kinetics to commercially sourced recombinant His-tagged human PD-1 and His-tagged cynomolgus PD-1 (Acro Biosystems).
Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were expressed with either a modified human IgG1 or a modified IgG4 isotype with a kappa light chain framework. Additional substitutions L235E, or L235A/G237A (LAGA, as described in Int'l Pub. No. WO1998/006248) (numbering based upon the EU numbering system) were introduced to the Fc region to abrogate effector functions of the immunoglobulin component.
The association constants (ka), dissociation constants (kd), and equilibrium constants (KD) of various anti-hPD-1 antibodies and anti-hPD-1 antibody-attenuated hIL-2 fusion proteins binding to recombinant human or cynomolgus PD-1 proteins was determined from the titration curves and the Carterra Kinetics software. The maximal feasible SPR signal generated (Rmax) and residual standard deviation (Res SD) was also calculated. The results from the kinetics screen are summarized in Table 21, and demonstrated that the addition of the attenuated hIL-2 moiety on anti-hPD-1 antibodies did not modulate PD-1 antibody binding to the human PD-1 or cynomolgus PD-1 antigens. In a separate experiment, H7-632-hIgG1-LAGA (SEQ ID NOs: 414 and 415) was measured by SPR and had a steady state equilibrium dissociation constant (KD) of 1.23×10−9 M and H7-767 had a KD=1.93×10−9 M.
Anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins were assayed for competition with one another using a sandwich method. Antibodies and corresponding antibody-IL-2 cytokine-fusion proteins were immobilized to HC30M chips using amine coupling chemistry described in Example 13. Following kinetic analysis described in Example 13, 80 nM human PD-1 (Acro Biosystems, Cat #PD-1-H5221-100 ug) was injected into the whole array. Competing anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins (analyte) were diluted to g/mL and subsequently injected into the array and binding parameters were assessed using SPR. Assessment of all anti-hPD-1 and anti-hPD-1-hIL-2 fusion proteins was performed in duplicate. Some variants tested had a modified human IgG1 or IgG4 kappa light chain framework with additional L235E or L235A/G237A (LAGA) substitutions to abrogate effector function of the immunoglobulin.
The screening of pairs of anti-hPD-1 or anti-hPD-1-attenuated hIL-2 fusion proteins allowed the identification of two bins, shown in Table 22. Antibodies and fusion proteins from Group 1 were able to bind hPD-1 in the presence of all antibodies and fusion proteins from Group 2, but competed with all members of the same Group. Antibodies and fusion proteins from Group 2 were able to bind hPD-1 in the presence of all antibodies and fusion proteins from Group 1, but competed with all members of the same Group. None of the anti-hPD-1 listed in Group 1 in Table 22 competed with KEYTRUDA® and OPDIVO®.
Anti-hPD-1-attenuated hIL-2 fusion proteins were tested for antagonism of hPD-1. Characterization of anti-hPD-1-attenuated hIL-2 fusion proteins was performed according to General Methods Protocol C.
For competition assays using the cell-based co-culture assay described in General protocol C, a few modifications were performed. Samples of the anti-hPD-1-attenuated hIL-2 fusion proteins were diluted to a fixed concentration of 400 nM and 20 μL was added to 20 L of titrated anti-hPD-1 #1 or anti-hPD-1 #2. The 40 μL mixture was added to CHO cells. Forty (40) L of Jurkat PD-1 effector cells were overlayed on the mixture of CHO cells and anti-hPD-1-attenuated hIL-2 fusion proteins. In this competition assay, a final concentration of saturating 100 nM anti-hPD-1-attenuated hIL-2 fusion proteins was tested in combination with titrated anti-hPD-1 #1 or anti-hPD-1 #2. The rest of the assay was performed as described in General Protocol C.
In the converse experiment, either anti-hPD-1 #1 or anti-hPD-1 #2 were diluted to a concentration of 400 nM and 20 μL was combined with 20 μL of titrated anti-hPD-1-attenuated hIL-2 fusion proteins. Anti-hPD-1-attenuated hIL-2 fusion proteins were serially titrated and the 40 μL mixture was added to CHO cells, then overlayed with 40 μL of Jurkat PD-1 Effector cells. The rest of the assay was performed as described in General Protocol C.
Anti-hPD-1-attenuated hIL-2 fusion proteins were evaluated for the level of attenuation of hIL-2 activity using the cell proliferation assays on NK-92 and TF1+IL-2Rβ cell lines as described in General Protocol E. Control fusion proteins included fusion proteins incorporating an anti-DNase I antibody (designated 1H3) with a human IgG4 or human IgG1 backbone directly fused to hIL-2 or with a linker (SEQ ID NO: 355) to demonstrate the effects of non-targeting attenuated hIL-2 fusion proteins. The hIL-2 sequence of these constructs contained substitutions for attenuated hIL-2 activity as described in Example 2. Full, partial, or no agonistic IL-2 activity (inactive) was also assessed similarly to Example 3. Some of the variants tested were expressed on a modified human IgG1 or IgG4 isotype with a kappa light chain, with additional L235E or L235A/G237A (LAGA) substitutions in the Fc region to abrogate immunoglobulin effector function. In some antibody-cytokine fusion proteins, the hIL-2 cytokine was fused to the C-terminus of the light chain (LC fusion).
The calculated EC50 of each antibody-cytokine fusion protein was determined from relative luminescence units (RLU), and fold change EC50 was calculated when compared with recombinant human IL-2 (rhIL-2). The fold change from rhIL-2 and agonistic activity is summarized in Table 23. Agonistic activity was measured as full, partial, or inactive as determined by the maximal luminescence of antibody-attenuated hIL-2 fusion proteins in comparison to the maximal luminescence of rhIL-2. Antibody-attenuated hIL-2 fusion proteins dose-titration curves that reached the maximal luminescence as the rhIL-2 were considered to be variants with full activity. Partial activity was calculated as a percentage of full activity using rhIL-2 maximal luminescence as 100%. Maximal RLU of antibody-attenuated hIL-2 fusion proteins with less than 10% of the rhIL-2 maximal RLU at the highest concentration of 1200 nM were considered to have no agonist activity or inactive. For some variants EC50 values were estimated only since maximal luminescence was not reached, as annotated by an a in Table 23.
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a Fold change is an estimate only since a full four parameter logistic curve was not reached
Anti-hPD-1-attenuated hIL-2 fusion proteins were evaluated for rescue of hIL-2 activity using a targeted cell line expressing hPD-1. Briefly, the TF1+IL-2Rβ cell line described in General Methods Protocol D was modified through lentiviral transduction to express the hPD-1 receptor (SEQ ID NO: 580). Flow cytometry with a Brilliant Blue 515 conjugated hPD-1 antibody (BD Biosciences Cat #565936) was used to detect hPD-1 expressing TF1+IL-2Rβ cells. Cells were sorted for low hPD-1 expression (less than 103 intensity on the Brilliant Blue 515 fluorophore). The pool was sorted twice more to collect cells that approximated hPD-1 expression levels on activated primary cells. This cell line (TF1+IL-2Rβ+hPD-1) was expanded and frozen in aliquots for the cell-based proliferation assays. Proliferation assays were performed as described in General Methods Protocol E with an incubation period of 3 days. Some variants tested had a modified human IgG1 or IgG4 kappa light chain framework with additional L235E or L235A/G237A (LAGA) substitutions to abrogate effector function of the immunoglobulin.
Table 24 summarizes the results from the proliferation assays on the targeted TF1+IL-2Rβ+hPD-1 cell line. Agonistic activity was measured as full, partial, or inactive as determined by the maximal luminescence of antibody-attenuated hIL-2 fusion proteins in comparison to the maximal luminescence of rhIL-2. Antibody-attenuated hIL-2 fusion protein dose-titration curves that reached the maximal luminescence as the rhIL-2 were considered to be variants with full activity. Partial activity was calculated as a percentage of full activity using rhIL-2 maximal luminescence as 100%. Maximal RLU of antibody-attenuated hIL-2 fusion proteins with less than 10% of the rhIL-2 maximal RLU at the highest concentration of 1200 nM were considered to have no agonist activity or inactive. For some variants, EC50 values were estimates only since a full curve was not reached. Many examples of anti-hPD-1-hIL-2 fusion proteins with attenuated hIL-2 showed rescued hIL-2 activity on the targeted cell line where the non-targeting antibody controls (denoted with 1H3) demonstrated no rescue of hIL-2 activity. Full rescue was illustrated by the reduction of fold-change from rhIL-2 to a value of 0 or 1.
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a Fold change is an estimate only since a full four parameter logistic curve was not reached
Since there are no accepted models to explore in vivo efficacy of oncology therapeutics in primates, a surrogate anti-mPD-1-attenuated hIL-2 fusion protein was generated and tested in a syngeneic murine tumor model. This MC38 colon adenocarcinoma model is routinely used to test efficacy of immuno-oncology therapeutics. To explore the in vivo effect of the anti-PD-1-attenuated hIL-2 fusion protein, a surrogate anti-mouse PD-1 antibody designated RMP1-14 (known to block mouse PD-L1 binding) and RMP1-30 (described as a mouse PD-L1 non-blocker) was fused to an attenuated hIL-2 at the C-terminus of the mouse IgG2b-N297A heavy chain and tested in an MC38 colon adenocarcinoma model. The hIL-2 moiety included the substitutions F42K, Y45R, and V69R that were tested on an IL-2 dependent mouse T lymphoblast cell line (CTLL-2) and that were demonstrated to be attenuated for mouse IL-2 activity. Human IL-2 can stimulate proliferation of mouse T cells at similar concentrations, however the same substitutions that attenuate activity on human IL-2 dependent cell lines do not attenuate activity on the CTLL-2 cell line (data not shown). As such, the F42K/Y45R/V69R substitutions were used in hIL-2 as a surrogate since they demonstrated attenuated IL-2 activity on mouse cell lines. Sequences comprising the heavy and light chain variable region sequences of anti-mouse PD-1 antibodies RMP1-14 and RMP1-30 (as described in Matsumoto K et al., J Immunol. 2004 Feb. 15; 172(4):2530-41) were also formatted onto a murine IgG2b-N297A background to generate anti-mPD-1 RMP1-14 mIgG2b-N297A (SEQ ID NOs: 564 and 566) and anti-mPD-1 RMP1-30 mIgG2b-N297A (SEQ ID NOs: 567 and 568). The mouse IgG2b isotype with an N297A substitution is the murine equivalent of an Fc isotype that abrogates Fc immune effector function. Surrogate antibodies and antibody-attenuated hIL-2 fusion proteins were produced, expressed and Protein-A purified using standard techniques.
In this murine tumor model, ten week old female C57BL/6NCrl (Charles River) mice were injected into the right flank with 5×105 MC38 colorectal carcinoma cells. When tumors reached 80-120 mm3, mice were sorted into cohorts (10 mice/group) and treatment began on day 1 of study. Anti-mPD-1 RMP1-14 mIgG2b-N297A, anti-mPD-1 RMP1-30 mIgG2b-N297A, anti-mPD-1 RMP1-14 mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R) (SEQ ID NOs: 565 and 566), and anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R) (SEQ ID NOs: 568 and 569) were dosed intraperitoneally at 5 mg/kg twice weekly for 4 weeks along with vehicle control (phosphate-buffered saline). Tumor size was measured with calipers twice weekly using the formula (w2×L)/2 where w=width and L=length for the duration of the study. The study endpoint was a tumor volume of 1000 mm3 or survival at day 50, whichever came first.
To understand the mechanism-of-action of the surrogate anti-hPD-1-attenuated hIL-2 fusion protein in vivo, a similar in vivo experiment to Example 18 was performed, followed by immunophenotyping of the resultant T cell populations in tumors, blood, spleens and lymph nodes after three doses. Ten week old female C57BL/6NCrl (Charles River) mice were subcutaneously implanted with the 5×105 murine MC38 colon adenocarcinoma cancer tumor cells into the right flank and tumors were monitored for growth. Animals with tumors between 150-260 mm3 were divided between four groups with 10 mice per group for the study. After 21 days post-implantation, animals were dosed intraperitoneally with 0.2 mL/dose phosphate buffered saline (PBS) for the vehicle control, 5 mg/kg anti-KLH-C3-mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R), 5 mg/kg anti-mPD-1 RMP1-30 mIgG2b-N297A, or 5 mg/kg anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R) on days 1, 4 and 8. On day 9, tumors, spleens and inguinal lymph nodes were harvested from all mice and processed into single cell suspensions for subsequent flow cytometry analysis.
The expansion of CD8+ T Effector Memory and decrease in Regulatory T cells has been associated with effective immunotherapy in both mice and humans.
Engrafting human immune cells into NOD-Prkdcem26Cd52IL-2rgem26Cd22/NjuCrl (NCG) mice that lack functional T, B, and NK cells has been a valuable tool for evaluating efficacy of therapeutics hypothesized to stimulate human T cells. In this model, if the therapeutic activates human T cells, there would be a resulting expansion of T cells and accelerated graft-versus-host disease (GvHD).
Three independent donors for human peripheral mononuclear cell (hPBMC) engraftment were evaluated over a 4 week period for engraftment kinetics as well as expression of human PD-1 and human IL-2 receptors on T cells. Of the three donors tested, the donor that induced the most T cells with an intermediate window for GvHD was chosen. 1.5×107 hPBMCs were intravenously injected into NCG mice and divided into 8 groups of 8-16 mice. On days 7, 10, and 14, mice were intraperitoneally injected with three doses of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) (SEQ ID NOs: 471, 425) (2.5 mg/kg, 5 mg/kg, or 10 mg/kg), 1H3-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) (SEQ ID NOs: 546, 374) (5 mg/kg or 10 mg/kg), 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A) (SEQ ID NOs: 563, 374) (10 mg/kg), or 2H7-hIgG1-LAGA-df-hIL-2 (T3A/R38E/I92K/C125A) (SEQ ID NOs: 474, 425) (5 mg/kg). The anti-DNase fusion protein both as a wild-type hIL-2 (1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A)) and with the attenuated hIL-2 moiety (1H3-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A)) was used as a non-targeting antibody control. Although the 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A) fusion protein had no changes in the hIL-2 moiety which reduce hIL-2 activity, it did comprise the T3A and C125A substitutions to remove the predicted O-linked glycosylation site on human IL-2 (see for example Int'l Pub. No. WO2012/107417) and unpaired cysteine residue (see for example Int'l Pub. No. WO2018/184964), respectively. These substitutions have not demonstrated reduced hIL-2 potency in the clinic. On Day 21, blood, spleen, and lungs were harvested in which blood and spleens were processed for flow cytometry immunophenotyping while lungs were weighed.
After 21 days, flow cytometry immunophenotyping was performed on the blood and spleens of animals. Table 25 summarizes the markers used to delineate human T cell populations for subsequent analysis.
Body weight was measured for 21 days and normalized to day 1 for each individual animal as an assessment of graft-versus-host disease (GvHD) as illustrated in
The flow cytometry analysis correlated with the accelerated graft-versus-host disease (GvHD) observed. Using the phenotypic markers for human T cell subset delineation provided in Table 25, flow cytometry analysis of peripheral blood demonstrated only a minor expansion of CD3+, CD4+, and CD8+ T cell subsets (as quantified by a fold change from vehicle control of between 10-fold to 50-fold for CD3+ T cells) in mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) at 2.5 mg/kg and 5 mg/kg, and mice treated with 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A) at 10 mg/kg. Furthermore, CD3+, CD4+, and CD8+ T cell subsets were greatly expanded (fold change from vehicle control was greater than 50-fold for CD3+ T cells) in the peripheral blood of mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) at 10 mg/kg. Table 26 summarizes the expanded human T cell subsets.
In addition to evaluating CD3+, CD4+, and CD8+ T cells between treatment groups, the memory and naïve subsets for CD4+ and CD8+ T cell subsets were also assessed. The phenotypic markers used for delineation of Naïve, Effector, Effector Memory and Central Memory for both CD4+ and CD8+ T cell is summarized in Table 25. There were no changes in Naïve, Effector or Central Memory T cells between treatment groups (data not shown). However, mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) at 10 mg/kg had greatly expanded CD4+ and CD8+ Effector Memory (EM) T cells in the peripheral blood with an average cell number per milliliter greater than 5 million for CD8+ T cells and greater than 50 million for CD4+ T cells (
In addition to stimulating effector T cells, IL-2 has been described to stimulate NK cells and regulatory T cells (Tregs) and since Tregs express high levels of CD25 and NK cells express CD122, these immune cell types were also evaluated.
Cynomolgus monkeys previously have been used to evaluate the toxicity of unmodified IL-2. Lethality was observed in cynomolgus monkeys at exogenous recombinant IL-2 doses as low as 50 μg/kg/day. Since the binding of H7-767 to cynomolgus monkey hPD-1 on primary activated PBMCs was confirmed by flow cytometry (Example 12), a single-dose study for preliminary safety assessment was performed with both a variant of H7-767 (H7-02-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) (SEQ ID NOs: 582 and 583) and H7-767. H7-02-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) was delivered by 15 minute iv infusion to 8 monkeys at 1 mg/kg (4 animals) or 10 mg/kg (4 animals). Sampling at time-points up to 360 hours following infusion was performed. No adverse effects, gross toxicities, body weight loss, or lethality was observed (data not shown). A follow-up single-dose study using H7-767 was performed at higher doses of 5 mg/kg and 50 mg/kg similar to the first study, with sampling at time-points up to 360 hours post-infusion. Again, no adverse effects, gross toxicities, body weight loss or lethality was observed (data not shown).
The attenuation of IL-2 activity of modified hIL-2 proteins comprising a substitution at amino acid position 20 (D20) and a substitution at amino acid position 38 (R38) was tested in proliferation assays in both the NK-92 and TF1+IL-2Rβ cell lines as described in Example 5 above. The modified hIL-2 proteins were grouped into 7 groups (1 to 7) based upon the maximal agonist activity of the modified hIL-2 protein and the level of attenuation of potency on both the intermediate and high-affinity receptors (Table 27) relative to non-modified recombinant hIL-2. The criteria used for grouping the modified hIL-2 proteins was:
Ten week old female C57BL/6NCrl mice were injected into the right flank with 5×105 syngeneic MC38 colorectal carcinoma cells. When tumors reached 80-120 mm3, mice were sorted into cohorts (10 mice/group) and treatment began on day 1 of study. All agents except hIL-2 were dosed intraperitoneally at 5 mg/kg twice weekly for 4 weeks, starting on day 1. hIL-2 was dosed intraperitoneally at 36,000 International Units once a day from days 1-5. Tumor size was measured with calipers twice weekly for the duration of the study. The study endpoint was a tumor volume of 1000 mm3 or survival at day 50 or progression free survival at day 70, whichever came first.
All test agents including antibody molecules and antibody-hIL-2 fusion proteins were generated using a mouse IgG2b Fc region with a single N297A amino-acid substitution at position 297, which prevents glycosylation of the Fc region and significantly reduces any Fc region-mediated immune effector function, thereby preventing cellular depletion in vivo. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (Matsumoto, J Immunol 172: 2530-2541, 2004). Anti-mPD-1 RMP1-14-hIL-2 F42K/Y45R/V69R is a bi-functional fusion protein consisting of the monoclonal RMP1-14 antibody antagonist of the mouse PD-1 receptor fused at its C-terminus via a flexible six amino-acid glycine/serine linker to hIL-2 F42K/Y45R/V69R (SEQ ID NO: 621) that is a reduced potency IL-2 variant. This molecule was designed to target a reduced potency hIL-2 variant directly to PD-1 expressing T cells in vivo in mice. Anti-KLH-hIL-2 F42K, Y45R, V69R is a control fusion protein consisting of an isotype control monoclonal antibody recognizing a non-mammalian antigen (keyhole limpet hemocyanin, KLH) fused at its C-terminus via a flexible six amino-acid glycine/serine linker to hIL-2 F42K, Y45R, V69R that is a reduced potency IL-2 variant.
Results are presented in
Mice that had undergone a complete tumor regression in the primary tumor study described in Example 23 and that had survived to day 50 were subjected to a secondary tumor challenge without any additional drug therapy. For tumor re-challenge, mice were implanted on the left flank contralateral to the location of the primary tumor with 5×105 MC38 tumor cells. As a control group, 10 age-matched tumor naïve mice were also implanted with MC38 tumor cells.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments disclosed herein and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.
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FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTRMLTHKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLALKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTDMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTRMLTHKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT
FMCEYADETATIVEFLNRWITFCQSIISTLT
PKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT
FMCEYADETATIVEFLNRWITFAQSIISTLT
PKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT
FMCEYADETATIVEFLNRWITFAQSIISTLT
YKNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS
ETTEMCEYADETATIVEFLNRWITFCQSIISTLT
YKNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS
ETTEMCEYADETATIVEFLNRWITFCQSIISTLT
YKNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNEHLRPRDLISNINVIVLELKGS
ETTEMCEYADETATIVEFLNRWITFAQSIISTLT
KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADETATIVEFLNRWITFCQSIISTL
T
CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADETATIVEFLNRWITECQ
SIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNEHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYA
DETATIVEFLNRWITFCQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
AKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
SKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
AKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
IKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
QKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
TKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
WKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVKVLELKGSETTEMCEYADE
TATIVEFLNRWITFCQSIISTLT
LTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEM
CEYADETATIVEFLNRWITFCQSIISTLT
LTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEM
CEYADETATIVEFLNRWITFAQSIISTLT
LTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEM
CEYADETATIVEFLNRWITFAQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTE
MCEYADETATIVEFLNRWITFCQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTE
MCEYADETATIVEFLNRWITFCQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTE
MCEYADETATIVEFLNRWITFAQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTE
MCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTE
MCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVKVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVKVLELKGSE
TTEMCEYADETATIVEFLNRWITFAQSIISTLT
KLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVKVLELKGSETTF
MCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTEMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRFLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVRVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVEVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVSVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVDVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
KNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFAQSIISTLT
YKNPKLTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS
ETTEMCEYADETATIVEFLNRWITFAQSIISTLT
TEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMC
EYADETATIVEFLNRWITFAQSIISTLT
LTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEM
CEYADETATIVEFLNRWITFAQSIISTLT
LTEMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEM
CEYADETATIVEFLNRWITFAQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFAQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFAQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFAQSIISTLT
FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNEHLRPRDLISNINVIVLELKGSETTEMCEYADE
TATIVEFLNRWITFAQSIISTLT
TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMC
EYADETATIVEFLNRWITFAQSIISTLT
QMILNGINNYKNPKLTRMLTKKERMPKKATELKHLQCLEEELKPLEERLNLAQSKNFHLRPRDLISNIN
VIVLELKGSETTEMCEYADETATIVEFLNRWITFCQSIISTLT
MILNGINNYKNPKLTRMLTKKFrMPKKATELKHLQCLEEELKPLEErLNLAQSKNFHLRPRDLISNINV
IVLELKGSETTEMCEYADETATIVEFLNRWITFCQSIISTLT
QMILNGINNYKNPKLTRMLTKKFrMPKKATELKHLQCLEEELKPLEErLNLAQSKNFHLRPRDLISNIN
VIVLELKGSETTEMCEYADETATIVEFLNRWITFCQSIISTLT
NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVL
ELKGSETTEMCEYADETATIVEFLNRWITFCQSIISTLT
The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.
This application claims priority to U.S. Provisional Application No. 63/352,842, which was filed on Jun. 16, 2022, U.S. Provisional Application No. 63/481,630, which was filed on Jan. 26, 2023, and U.S. Provisional Application No. 63/502,746, which was filed on May 17, 2023, the disclosure of each of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63352842 | Jun 2022 | US | |
| 63481630 | Jan 2023 | US | |
| 63502746 | May 2023 | US |
