TARGETED DELIVERY OF TUMOR MATRIX MODIFYING ENZYMES
20220363778 · 2022-11-17
Inventors
Cpc classification
C07K2317/73
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
C07K2317/569
CHEMISTRY; METALLURGY
A61K47/6855
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Provided are compositions and methods for treatment of tumors. The compositions comprises a fusion construct comprising single domain antibody (sdAb) that is specific for HER2, collagenase, and optionally, albumin binding domain. Methods are provided for increasing penetrability of tumors and inhibiting the growth of tumors comprising administering a fusion construct comprising anti-HER2 specific sdAb, collagenase, and optionally albumin binding domain, alone or in combination with and an anti-tumor agent.
Claims
1. A fusion protein comprising a collagenase and a single domain antibody (sdAb) which is specific for HER2.
2. The fusion protein of claim 1, wherein the sdAb has a sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:3 and the collagenase has a sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:5.
3. The fusion protein of claim 1, wherein the fusion protein comprises a sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:1.
4. The fusion protein of claim 1, wherein the sdAb binds to an epitope of HER2 that is distinct from the epitope targeted by trastuzumab, epitope targeted by pertuzumab, or both.
5. The fusion protein of claim 1, further comprising an albumin binding domain (ABD).
6. The fusion protein of claim 5, wherein the fusion protein comprises a sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:9 or SEQ ID NO:10.
7. The fusion protein of claim 1, wherein amino acid linkers are present between the amino acid sequences of sdAb and the collagenase, and collagenase and ABD.
8. A pharmaceutical composition comprising the fusion protein of claim 1.
9. A method of treating a tumor comprising administering to an individual in need of treatment a composition of claim 8.
10. The method of claim 9, wherein the individual is further administered an antibody directed to HER2, or an antibody-drug conjugate, wherein the antibody in the antibody-drug conjugate is directed to HER2.
11. The method of claim 10, wherein the composition is delivered at or near a tumor.
12. The method of claim 10, wherein the composition is delivered intratumorally.
13. The method of claim 9, wherein the composition is delivered intravenously.
14. The method of claim 10, wherein the antibody directed to HER2 is trastuzumab or pertuzumab.
15. The method of claim 10, wherein the antibody-drug conjugate is T-DM1.
16. The method of claim 10, wherein the fusion protein and the antibody or the antibody-drug conjugate are administered in the same composition.
17. The method of claim 10, wherein the fusion protein and the antibody or the antibody-drug conjugate are administered in separate compositions.
18. The method of claim 10, wherein the tumor is a solid tumor.
19. The method of claim 18, wherein the tumor is breast, pancreatic, gastric, intestinal, ovarian, colorectal, brain, lung, or liver.
20. The method of claim 18, wherein the tumor comprises a dense collagen-containing extracellular matrix.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE DISCLOSURE
[0017] The present disclosure provides fusion proteins comprising antibody fragments having improved tumor penetrability and/or facilitating penetrability of other anti-tumor agents. Compositions comprising such fusion proteins and methods of using same are also provided.
[0018] The term “treatment” as used herein refers to reduction or delay in one or more symptoms or features associated with the presence of the particular condition being treated Treatment does not necessarily mean complete cure and does not preclude relapse of the condition. Treatment may be carried out over a short period of time (days, weeks), or over a long period of time (months) or may be on a continuous basis (e.g., in the form of a maintenance therapy). Treatment may be continual or intermittent.
[0019] The term “therapeutically effective amount” as used herein is the amount sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The exact amount desired or required will vary depending on the mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art (such as a clinician) with the benefit of the present disclosure.
[0020] Where a range of values is provided in this disclosure, it should be understood that each intervening value, to the tenth of the value of the lower limit between the upper and lower limit of that range, and any other intervening value in that stated range is encompassed within the disclosure, unless clearly indicated otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges encompassed within the disclosure.
[0021] As used in this disclosure, the singular forms include the plural forms and vice versa unless the context clearly indicates otherwise.
[0022] The term “single domain antibody” (sdAb) is used interchangeably with the term “nanobody” to mean an antibody fragment representing a single monomeric variable antibody domain which is able to bind selectively to an antigen. A sdAb may comprise heavy chain variable domains or light chain variable domains. In an embodiment, the sdAb of the disclosure comprises heavy chain variable domain. A sdAb or nanobody may be derived from camelids (VHH fragments) or cartilaginous fishes (VNAR fragments), or may be derived from splitting the dimeric variable domains from IgG into monomers.
[0023] A reference to antibody derivatives and fragments in this disclosure includes any antigen binding fragment of an antibody or modification of the antibody. Examples include, but are not limited to, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, CDR fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, nanobodies, chimeric antibodies, and fusion proteins comprising any of the foregoing or comprising an antibody or ADC.
[0024] In an aspect, this disclosure provides a fusion protein (also referred to herein as a fusion construct) of collagenase and a single domain antibody that binds specifically to human epidermal growth factor receptor 2 (HER2). In an embodiment, the Kd value for the sdAb binding to HER2 may be less than 50 nM. In embodiments, the Kd may be less than 25, 10, 5, 1 nM, or less than 750, 500, or 100 pM. The collagenase may be from any source. In an embodiment, the collagenase is clostridial collagenase H. The sdAb may be a HER2 binding domain of an antibody that is specific for HER2. In an embodiment, the antibody binds to an epitope that is distinct from the epitope targeted by trastuzumab and therefore, does not compete with trastuzumab or TDM1 for HER2 binding. In an embodiment, the sdAb is 2Rs15d, a dromedary-derived sdAb first reported by Vaneycken et.al. 2011 (PMID: 21478264). In an embodiment, an anti-HER2 sdAb, 2Rb17c may be used instead of 2Rs15d.
[0025] The collagenase may be directly linked to the sdAb or indirectly linked to the sdAb via a linker. The fusion protein may comprise the C-terminal of collagenase linked directly to the N-terminal of the sdAb, or may comprise the C-terminal of the sdAb linked directly to the N-terminal of collagenase. In an embodiment, this disclosure provides a fusion protein comprising: collagenase, sdAb that binds specifically to HER2, and a linker linking the C-terminal of the sdAb to the N-terminal of the collagenase. In an embodiment, this disclosure provides a fusion protein comprising: collagenase, sdAb that binds specifically to HER2, and a linker linking the C-terminal of the collagenase to the N-terminal of the sdAb. Linking the sdAb to collagenase, either directly or via a linker, should not eliminate either the binding function of the sdAb or the enzymatic function of collagenase. Suitable linkers include amino acid chains and alkyl chains functionalized with reactive groups for coupling to both the nanobody and collagenase. An amino acid chain linker may be about 1 to about 40 amino acid residues, such as 1 to 10 amino acid residues. In an embodiment, the fusion protein may further comprise an albumin binding domain, which may be present at the N-terminal end, the C-terminal end, or between the sdAb and collagenase.
[0026] An advantage of fusing the single domain antibody to a matrix digesting enzyme is the tumor selectivity advantage provided through the sdAb that limits off-target exposure to active enzyme.
[0027] In an embodiment, the fusion protein of the present disclosure comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:1. In this fusion protein, amino acids 1 to 115 represent the sdAb (2Rs15d) and amino acids 131 to 976 represent collagenase (colH), and amino acids 116 to 130 represent the linker (glycine serine linker). A polyhistidine tag (hexahistidine tag) is also shown from amino acids 977 to 982. The disclosure also encompasses fusion proteins comprising amino acids 1 to 115 and 131 to 976 without the intervening linker or with a different linker. In an embodiment, this disclosure provides variants of the fusion protein, wherein a variant of the fusion protein is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:1. Any variant of the fusion protein of this disclosure should have the HER2 binding function as well as the collagenase function of the fused protein of SEQ ID NO:1.
[0028] In an embodiment, the fusion protein of the present disclosure has a sequence which comprises a sdAb that binds specifically to HER2, and collagenase, and optionally, a linker, wherein the sequence of the sdAb is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of amino acids 1 to 115 of SEQ ID NO:1, and wherein if the linker is present between the sdAb and collagenase, sequences may be, from N- to C-terminus, sdAb-linker-collagenase, or collagenase-linker-sdAb. In an embodiment, the fusion protein of the present disclosure has a sequence which comprises a sdAb that binds specifically to HER2, optionally a linker, and collagenase, wherein the sequence of the collagenase is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of amino acids 131 to 976 of SEQ ID NO:1, and wherein if the linker is present between the sdAb and collagenase, sequences may be, from N- to C-terminus, sdAb-linker-collagenase, or collagenase-linker-sdAb.
[0029] In an aspect, the disclosure provides a nucleic acid sequence encoding a fusion protein of SEQ ID NO:1 (or a variant thereof) as described herein. In an embodiment, the nucleic acid comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:2. In embodiments, the sequence which encodes a fusion protein or a variant thereof, as described herein may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence to SEQ ID NO:2.
[0030] The amino acid sequence of a fusion construct, termed herein as 2Rs15d-ColH is provided below as SEQ ID NO:1. In the sequence, the 2Rs15d (sdAb) sequence is not underlined, the glycine serine linker is italicized, the ColH (collagenase) sequence is underlined, and the hexahistidine tag is bolded.
TABLE-US-00001 (SEQ ID NO: 1) QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSR ISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYN LETYWGQGTQVTVSSGGGGSGGGGSGGGGSVQNESKRYTVSYLKTLNYYD LVDLLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEI DHKGIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNF KLGTEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKNIDRYALDDL KSKALFNVLAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYG KINDNNSWIIDNGIYHIAPLGKLHSNNKIGIETLTEVMKVYPYLSMQHLQ SADQIKRHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGA RVEEEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEE YKLNSVLYGYDTNNGGMYIEPEGTFFTYEREAQESTYTLEELFRHEYTHY LQGRYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVS NIHNTTRNNRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLN DLAKNNDVDGYDNYIRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADD YLVRHAYKNPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGAS KGKLEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYD VVFHGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVS YEWDFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKD LSENKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDF SSEQNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKE PNNSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGY GGATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMP YRINIELEHHHHHH.
[0031] In an embodiment, the fusion construct comprises collagenase, sdAb which is specific for HER2, and an albumin binding domain (ABD). The ABD, the sdAb, and the collagenase may be present in any configuration from the N- to the C-terminus. The construct may further comprise linkers between the sdAb, collagenase and ABD and amino acid sequences, such as polyhistines, may flank the N- or the C-terminal ends. For example, the configuration may be sdAb-linker-ColH-linker-ABD-hexahistidine, or ABD-linker-sdAb-linker-collagenase-hexahistidine.
[0032] An example of a sequence of a fusion construct comprising sdAb 2Rs15d, collagenase and ABD is shown in
TABLE-US-00002 (SEQ ID NO: 9) QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSR ISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYN LETYWGQGTQVTVSSGGGSGGGSGGGSVQNESKRYTVSYLKTLNYYDLVD LLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEIDHK GIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNFKLG TEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKNIDRYALDDLKSK ALFNVLAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGKIN DNNSWIIDNGIYHIAPLGKLHSNNKIGIETLTEVMKVYPYLSMQHLQSAD QIKRHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGARVE EEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEYKL NSVLYGYDTNNGGMYIEPEGTFFTYEREAQESTYTLEELFRHEYTHYLQG RYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSNIH NTTRNNRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLA KNNDVDGYDNYIRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADDYLV RHAYKNPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGASKGK LEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYDVVF HGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVSYEW DFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKDLSE NKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFSSE QNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPNN SKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYGGA TWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMPYRI NIEGGGSGGGSLEVLFQGPGGGSLAEAKVLANRELDKYGVSDFYKRLINK AKTVEGVEALKLHILAALPHHHHHH.
[0033] An example of a sequence of a fusion construct comprising sdAb 2Rb 17c, collagenase and ABD is shown in
TABLE-US-00003 (SEQ ID NO: 10) QVQLQESGGGLVQPGGSLRLSCAASGFIFSNDAMTWVRQAPGKGLEWVSS INWSGTHTNYADSVKGRFTISRDNAKRTLYLQMNSLKDEDTALYYCVTGY GVTKTPTGQGTQVTVSSGGGSGGGSGGGSVQNESKRYTVSYLKTLNYYDL VDLLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEID HKGIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNFK LGTEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKNIDRYALDDLK SKALFNVLAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGK INDNNSWIIDNGIYHIAPLGKLHSNNKIGIETLTEVMKVYPYLSMQHLQS ADQIKRHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGAR VEEEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEY KLNSVLYGYDTNNGGMYIEPEGTFFTYEREAQESTYTLEELFRHEYTHYL QGRYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSN IHNTTRNNRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLND LAKNNDVDGYDNYIRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADDY LVRHAYKNPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGASK GKLEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYDV VFHGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVSY EWDFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKDL SENKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFS SEQNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEP NNSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYG GATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMPY RINIEGGGSGGGSLEVLFQGPGGGSLAEAKVLANRELDKYGVSDFYKRLI NKAKTVEGVEALKLHILAALPHHHHHH.
[0034] In an embodiment, this disclosure provides variants of the fusion protein, wherein a variant of the fusion protein is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:9 or SEQ ID NO:10. Any variant of the fusion protein of this disclosure should have the HER2 binding function as well as the collagenase function.
[0035] An example of a nucleic acid sequence encoding a fusion construct of SEQ ID NO:1 is provided as SEQ ID NO:2, in which restriction enzyme site sequences are shown as bold and underlined, sequence encoding the 2Rs15d (sdAb) is neither underlined nor bolded, sequence encoding the glycine serine linker is italicized, and sequence encoding the ColH (collagenase) is underlined.
TABLE-US-00004 (SEQ ID NO: 2) catatgcaggttcagctgcaagaaagcggtggtggtagcgttcaggcagg cggtagcctgaaactgacctgtgcagcaagcggttatatctttaatagct gtggtatgggttggtatcgtcagagtccgggtcgtgaacgtgaactggtt agccgtattagcggtgatggtgatacctggcataaagaaagcgttaaagg tcgttttaccatcagccaggataacgtgaaaaaaaccctgtacctgcaga tgaatagtctgaaaccggaagataccgcagtgtatttttgtgccgtttgc tataatctggaaacctattggggtcagggcacccaggttaccgttagctc aggtggtggtggcagcggtggcggtggttctggtggcggaggtagcgtgc agaatgaaagcaaacgttataccgtgagctatctgaaaaccctgaactat tatgatctggttgatctgctggtgaaaaccgaaattgaaaatctgccgga cctgtttcagtatagcagtgatgcaaaagaattctacggtaataaaaccc gcatgagctttatcatggatgaaattggtcgtcgtgcaccgcagtataca gaaattgatcataaaggtattccgacgctggttgaagttgttcgtgcagg tttttatctgggctttcataacaaagaactgaacgagattaacaaacgca gctttaaagaacgtgtgattccgagcattctggccattcagaaaaatccg aactttaaactgggcaccgaagtgcaggataaaattgttagcgcaaccgg tctgctggcgggtaacgagaccgcgccgccggaagtggttaacaacttta ccccgattctgcaggactgcattaaaaacattgaccgttatgcgctggat gacctgaagagcaaagcgctgtttaacgttctggcggcgccgacctatga cattaccgagtatctgcgtgcgaccaaggagaaaccggaaaacaccccgt ggtacggcaaaatcgatggtttcattaacgagctgaaaaagctggcgctg tacggtaaaatcaacgacaacaacagctggatcattgacaacggtattta ccacatcgcgccgctgggcaaactgcacagcaacaacaagatcggcattg agaccctgaccgaagttatgaaggtgtacccgtatctgagcatgcaacac ctgcagagcgcggatcaaatcaaacgtcactacgatagcaaggacgcgga aggcaacaaaatcccgctggacaaattcaagaaagaaggcaaggagaaat actgcccgaaaacctatacctttgatgacggcaaggttattatcaaggcg ggtgcgcgtgtggaagaagagaaggtgaaacgtctgtattgggcgagcaa ggaagtgaacagccagttctttcgtgtttatggcattgataaaccgctgg aggaaggtaacccggatgacatcctgaccatggtgatctacaacagcccg gaagagtacaaactgaacagcgtgctgtacggctacgacaccaacaacgg tggcatgtacattgagccggaaggtacctttttcacctatgaacgtgagg cgcaggagagcacctataccctggaggagctgttccgtcacgagtatacc cactatctgcaaggtcgttatgcggtgccgggccagtggggtcgtaccaa actgtacgataacgaccgtctgacctggtatgaggaaggcggtgcggagc tgttcgcgggtagcacccgtaccagcggtattctgccgcgtaagagcatc gttagcaacattcacaacaccacccgtaacaaccgttacaagctgagcga caccgtgcacagcaagtatggcgcgagcttcgaattctacaactacgcgt gcatgttcatggactatatgtacaacaaggacatgggcattctgaacaaa ctgaacgacctggcgaagaacaacgatgttgacggttacgacaactacat tcgtgatctgagcagcaactatgcgctgaacgacaagtatcaggaccaca tgcaggagcgtattgacaactacgagaacctgaccgttccgtttgttgcg gacgattacctggttcgtcacgcgtacaagaacccgaacgaaatttatag cgaaatcagcgaggtggcgaaactgaaggatgcgaaaagcgaggttaaaa agagccaatacttcagcaccttcaccctgcgtggtagctataccggcggc gcgagcaagggcaaactggaggaccagaaagcgatgaacaagttcatcga cgatagcctgaagaagctggacacctatagctggagcggttacaaaaccc tgaccgcgtacttcaccaactataaagttgacagcagcaaccgtgtgacc tatgacgttgtgtttcacggttatctgccgaacgaaggcgatagcaagaa cagcctgccgtatggtaaaatcaacggcacctacaagggtaccgaaaagg agaaaatcaagtttagcagcgaaggtagcttcgacccggatggcaaaatc gtgagctacgaatgggattttggtgacggtaacaaaagcaacgaagaaaa cccggaacacagctacgataaagttggtacctacaccgtgaaactgaaag tgaccgatgacaagggcgaaagcagcgttagcaccaccaccgcggagatc aaagatctgagcgagaacaaactgccggtgatctacatgcacgttccgaa aagcggtgcgctgaaccagaaagttgtgttctatggcaaaggcacctacg atccggacggtagcatcgcgggctaccagtgggacttcggcgatggcagc gattttagcagcgagcagaacccgagccacgtgtataccaagaaaggcga atataccgtgaccctgcgtgttatggacagcagcggtcagatgagcgaga agaccatgaaaatcaagattaccgatccggtttacccgattggtaccgag aaggaaccgaacaacagcaaggaaaccgcgagcggtccgattgtgccggg tattccggttagcggcaccatcgagaacaccagcgaccaagattattttt acttcgatgttatcaccccgggcgaggtgaagatcgatattaacaaactg ggttacggttacggtggcgcgacctgggtggtttatgacgagaacaacaa cgcggttagctacgcgaccgacgatggccagaacctgagcggcaagttta aagcggataagccgggccgttactacatccacctgtatatgtttaacggt agctacatgccgtaccgtatcaacattgagctcgag.
[0036] The amino acid sequence of sdAb 2Rs15d is also provided:
TABLE-US-00005 (SEQ ID NO: 3) QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSR ISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYN LETYWGQGTQVTVSS.
[0037] The amino acid sequence of another anti-HER2 sdAb 2Rb17c is:
TABLE-US-00006 (SEQ ID NO: 4) QVQLQESGGGLVQPGGSLRLSCAASGFIFSNDAMTWVRQAPGKGLEWVSS INWSGTHTNYADSVKGRFTISRDNAKRTLYLQMNSLKDEDTALYYCVTGY GVTKTPTGQGTQVTVSS:
[0038] The amino acid sequence of Clostridium collagenase H is:
TABLE-US-00007 (SEQ ID NO: 5) VQNESKRYTVSYLKTLNYYDLVDLLVKTEIENLPDLFQYSSDAKEFYGNK TRMSFIMDEIGRRAPQYTEIDHKGIPTLVEVVRAGFYLGFHNKELNEINK RSFKERVIPSILAIQKNPNFKLGTEVQDKIVSATGLLAGNETAPPEVVNN FTPILQDCIKNIDRYALDDLKSKALFNVLAAPTYDITEYLRATKEKPENT PWYGKIDGFINELKKLALYGKINDNNSWIIDNGIYHIAPLGKLHSNNKIG IETLTEVMKVYPYLSMQHLQSADQIKRHYDSKDAEGNKIPLDKFKKEGKE KYCPKTYTFDDGKVIIKAGARVEEEKVKRLYWASKEVNSQFFRVYGIDKP LEEGNPDDILTMVIYNSPEEYKLNSVLYGYDTNNGGMYIEPEGTFFTYER EAQESTYTLEELFRHEYTHYLQGRYAVPGQWGRTKLYDNDRLTWYEEGGA ELFAGSTRTSGILPRKSIVSNIHNTTRNNRYKLSDTVHSKYGASFEFYNY ACMFMDYMYNKDMGILNKLNDLAKNNDVDGYDNYIRDLSSNYALNDKYQD HMQERIDNYENLTVPFVADDYLVRHAYKNPNEIYSEISEVAKLKDAKSEV KKSQYFSTFTLRGSYTGGASKGKLEDQKAMNKFIDDSLKKLDTYSWSGYK TLTAYFTNYKVDSSNRVTYDVVFHGYLPNEGDSKNSLPYGKINGTYKGTE KEKIKFSSEGSFDPDGKIVSYEWDFGDGNKSNEENPEHSYDKVGTYTVKL KVTDDKGESSVSTTTAEIKDLSENKLPVIYMHVPKSGALNQKVVFYGKGT YDPDGSIAGYQWDFGDGSDFSSEQNPSHVYTKKGEYTVTLRVMDSSGQMS EKTMKIKITDPVYPIGTEKEPNNSKETASGPIVPGIPVSGTIENTSDQDY FYFDVITPGEVK.
[0039] The amino acid sequence for a glycine serine linker is:
TABLE-US-00008 (SEQ ID NO: 6) GGGSGGGSGGGS.
[0040] The amino acid sequence for another linker is:
TABLE-US-00009 (SEQ ID NO: 7) GGGSGGGSLEVLFQGPGGGS
[0041] The amino acid sequence for ABD035 is:
TABLE-US-00010 (SEQ ID NO: 8) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP
[0042] The disclosure of a sequence in this disclosure with hexahistidines is intended to include a sequence of the construct without the hexahistidine also.
[0043] In an aspect, the present disclosure provides an expression vector comprising a sdAb-collagenase fusion construct of the disclosure. The expression vector is not particularly limiting other than by a requirement for the sdAb-collagenase fusion protein expression to be driven from a suitable promoter. Many suitable expression vectors and systems are commercially available. Examples of vectors include plasmids, cosmids, transposable elements, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). The expression vectors may be configured to produce fusion proteins. The fusion proteins may include components that facilitate purification, such as HIS or FLAG tag or improve solubility or secretion or other functions. The vector may have a high copy number, an intermediate copy number, or a low copy number. Expression vectors typically contain one or more of the following elements: promoters, terminators, ribosomal binding sites, and IRES. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid. A nucleic acid encoding a nanobody construct may also be operably linked to a nucleotide sequence encoding a selectable marker. A selectable marker may be used to efficiently select and identify cells that have integrated the exogenous nucleic acids. Selectable markers give the cell receiving the exogenous nucleic acid a selection advantage, such as resistance towards a certain toxin or antibiotic. Suitable examples of antibiotic resistance markers include those coding for proteins that impart resistance to kanamycin, streptomycin, spectinomycin, neomycin, gentamycin (G418), ampicillin, tetracycline, chloramphenicol, puromycin, hygromycin, zeocin, and blasticidin. An expression vector encoding a nanobody construct may be delivered to a host cell using a viral vector or via a non-viral method of transfer. Viral vectors suitable for introducing nucleic acids into cells include retroviruses, adenoviruses, adeno-associated viruses, rhabdoviruses, and herpes viruses. Non-viral methods of nucleic acid transfer include naked nucleic acid, liposomes, and protein/nucleic acid conjugates. An expression construct encoding a nanobody construct may be introduced into the cell by transfection. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. In an embodiment, the expression system is a bacterial expression system, involving, for example, E. coli. Host cells may be transfected with a vector comprising a nanobody construct and then cultured so that they transcribe and translate the desired polypeptide. The host cells may then be lysed to extract the expressed polypeptide for subsequent purification.
[0044] In an aspect, this disclosure provides host cells containing vector constructs as described herein. Host cells may contain nucleotide sequences that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be such that it modulates the expression of the inserted gene sequences, or modifies and processes the gene product as desired. Expression from certain promoters may be modified by the presence of certain inducers thereby allowing the expression of the genetically engineered polypeptide to be controlled. In an embodiment, the disclosure provides a fusion protein of a sdAb which is specific for HER2 and a matrix modifying enzyme, and optionally albumin binding domain. The matrix modifying enzyme may be collagenase. In an embodiment, the sdAb binds to an epitope that is distinct from the epitope targeted by trastuzumab (Herceptin™) and, therefore, the sdAb would not compete with trastuzumab or TDM1 for HER2 binding. In another embodiment, the sdAb binds to an epitope that is distinct from the epitope targeted by pertuzumab (Perjeta). In an embodiment, the sdAb binds to an epitope that is distinct from the epitope targeted by either trastuzumab or pertuzumab.
[0045] In an aspect, the disclosure provides pharmaceutical compositions comprising the fusion protein as described herein. The formulations typically contain physiologically acceptable carriers, excipients or stabilizers and may be in the form of aqueous solutions, lyophilized or other dried or solid formulations. Examples of suitable pharmaceutical preparation components can be found in Remington: The Science and Practice of Pharmacy 20th edition (2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, polyethylene glycol (PEG) and the like.
[0046] In an aspect, this disclosure provides a method for improving penetrability of an antitumor antigen antibody or a fragment thereof by administering to an individual in need of treatment a composition comprising the antibody or an antigen binding fragment thereof fused to a matrix modifying enzyme, such as collagenase, and optionally further comprising albumin binding domain. In an embodiment, the disclosure provides a method for inhibiting the growth of or proliferation of a tumor comprising administering to an individual in need of treatment anti-tumor agent, and an sdAb specific for a tumor antigen fused to a matrix modifying enzyme, such as collagenase, with the fusion protein optionally further comprising an albumin binding domain. In an embodiment, the disclosure provides a method for treatment of HER2+ tumors comprising administering to an individual who is afflicted with a HER2+ tumor, a composition comprising a HER2 specific sdAb fused to collagenase—either directly or via a linker, and optionally further comprising albumin binding domain in the fusion protein, whereby the penetrability of the fusion protein within the tumor is greater than the penetrability of the sdAb alone.
[0047] In an embodiment, the disclosure provides a method for improving the penetrability and distribution of an anti-tumor agent (such as an antibody, antibody derivative or fragment, antibody drug conjugate, anti-tumor macromolecule, anti-tumor molecule) within a tumor comprising administering to an individual in need of treatment the anti-tumor agent and a fusion protein comprising an antitumor antigen antibody or a fragment or derivative thereof and collagenase, and optionally an albumin binding domain. The anti-tumor agent and the anti-tumor antibody (or fragment or derivative thereof) may be the same or different. The anti-tumor agent and the fusion protein may be administered in the same composition or as separate compositions. When administered as separate compositions, they may be administered at the same time or different times, same route or different routes, over the same period of time or different periods of time, which may overlap. As an example, the disclosure provides a method for improving the distribution of trastuzumab or T-DM1 within a tumor comprising administering to the individual (and the tumor), the trastuzumab or T-DM1 and a fusion protein comprising anti-HER2 antibody or a fragment or derivative thereof, such as a sdAb (e.g., 2Rs15d), wherein the growth of the tumor is inhibited more than if the trastuzumab or T-DM1 is administered without the fusion protein. In an example, the disclosure provides a method for improving the distribution of pertuzumab within a tumor comprising administering to the individual (and the tumor), pertuzumab and a fusion protein comprising anti-HER2 antibody or a fragment or derivative thereof, such as a sdAb (e.g., 2Rs15d), wherein the growth of the tumor is inhibited more than if the pertuzumab is administered without the fusion protein.
[0048] In an embodiment, the fusion construct of the present disclosure improves the distribution of an anti-tumor agent that is administered in combination with the fusion construct. The anti-tumor agent may be an anti-tumor antigen antibody, an antibody derivative, antibody fragment or any macromolecule having anti-tumor growth activity. Antibody derivatives and fragments include, but are not limited to Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, CDR fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, nanobodies, chimeric antibodies, and fusion proteins comprising any of the foregoing or comprising an antibody or ADC.
[0049] In an embodiment, the disclosure provides a method for improving penetrability of an antitumor antibody or a conjugate of the antibody (such as an antibody-drug conjugate (ADC)) comprising administering to an individual in need of treatment, i) a fusion protein comprising a tumor antigen specific sdAb, tumor matrix modifying enzyme, and optionally, albumin binding domain, and ii) the antitumor antibody or a conjugate of the antibody (such as an ADC). In an embodiment, the method comprises administering to an individual in need of treatment, i) a fusion protein comprising a HER2 specific sdAb, collagenase, and optionally, an albumin binding domain; and ii) anti-HER2 antibody or an ADC comprising an anti-HER2 antibody. In an embodiment, the method comprises administering to an individual in need of treatment, i) a fusion protein comprising a HER2 specific sdAb (such 2Rs15d), collagenase, and optionally, an albumin binding domain; and ii) trastuzumab or T-DM1, or pertuzumab.
[0050] In an embodiment, the disclosure provides a method for inhibiting the growth of or proliferation of tumor cells by administering to an individual who is afflicted with the tumor a fusion protein comprising a sdAb that is specific for HER2, and collagenase, wherein the fusion protein penetrates further into the tumor than the sdAb without being fused to collagenase, and/or exhibits increased inhibition of tumor cell growth compared to sdAb without fusion to collagenase. In an embodiment, the fusion protein as administered in combination with an anti-tumor agent, such as an anti-tumor antibody, such as sdAb, wherein the fusion protein facilitates the penetration of accompanying anti-tumor agent, (e.g., sdAb).
[0051] A composition comprising the fusion protein may be administered using any suitable route including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intratumoral administration, or at or near the tumor. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In an embodiment, the fusion protein is delivered intra-tumorally. The administration may be carried out in a continuous manner or may be intermittent. Appropriate dosage will depend upon the particular tumor being treated, the specifics and condition of the individual patient, the mode of administration etc. Determination of appropriate dosage is within the purview of one skilled in the art, such as a treating physician. In an embodiment, the amount of sdAb-collagenase fusion protein may be administered is from about 0.01 mg/kg to 500 mg/kg, or 0.1 mg/kg to about 100 mg/kg. In embodiments, the amount of fusion protein administered may be 0.1 mg/kg to about 50 mg/kg, or 0.1 mg/kg to about 25, 10, 5 or 1 mg/kg. In embodiments, the administered amount of the fusion protein may be 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 75.0, 100.0, 200.0, or 500.0 mg/kg.
[0052] The fusion constructs and the antibody or sdAb or antibody conjugates (such as ADCs) may be administered in the same composition or in different compositions, at the same time or at different times, by the same route or different routes, over the same period of time or different periods of time.
[0053] The fusion constructs of the present disclosure, such as sdAb-collagenase-ABD fusion protein, may be administered alone or in combination with other types of treatments (e.g., surgical resection, radiation therapy, chemotherapy, hormonal therapy, immunotherapy or other anti-tumor agents). Similarly, the fusion constructs and the antibody or sdAb or antibody conjugates (such as ADCs) may be administered in combination with other types of treatments (e.g., surgical resection, radiation therapy, chemotherapy, hormonal therapy, immunotherapy or other anti-tumor agents).
[0054] The present compositions may be used for any type of cancer, including carcinoma, lymphoma, sarcoma, melanoma and leukemia. Non-limiting examples include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, myeloma (including multiple myeloma), hepatocellular cancer, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma/glioma (e.g., anaplastic astrocytoma, glioblastoma multiforme, anaplastic oligodendroglioma, anaplastic oligodendroastrocytoma), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, brain cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. In an embodiment, the cancer is HER2+.In an embodiment, the tumor comprises dense collagen-containing extracellular matrix.
[0055] The present compositions may be particularly useful for patients where the HER2+ tumors, such as, for example, breast tumors, are found to be non-responsive to current treatments, such as trastuzumab, pertuzumab, and T-DM1. Alternatively, the present compositions may be used in combination with trastuzumab, pertuzumab, and/or T-DM1.
[0056] In an aspect, this disclosure provides kits for the treatment of cancer. The kit may comprise an anti-tumor agent and a fusion construct of the present disclosure. For example, a kit may comprise, in separate containers, trastuzumab, pertuzumab, and/or T-DM1, a fusion construct comprising sdAb directed to HER2, collagenase, and optionally albumin binding domain, and optionally instructions for use, which may include dosage and administration instructions. In an embodiment, the kit comprises in separate containers: i) trastuzumab, pertuzumab, and/or T-DM1, and 2) sdAb 2Rs15d. Multiple doses of the components may be provided.
[0057] The following are some non-restrictive examples of embodiments of the present disclosure.
[0058] Example 1. A fusion protein of a single domain antibody (sdAb) and collagenase.
[0059] Example 2. A fusion protein of a single domain antibody (sdAb) which is specific for HER2, and collagenase, wherein the sdAb and the collagenase are covalently linked.
[0060] Example 3. The fusion protein of Example 1 or Example 2, wherein the sdAb and the collagenase are covalently linked via a linker.
[0061] Example 4. A fusion protein having the sequence as set forth in SEQ ID NO:1.
[0062] Example 5. A pharmaceutical composition comprising the fusion protein of any one of Examples 1-4.
[0063] Example 6. A method of treating a tumor comprising administering to an individual in need of treatment a composition of Example 5.
[0064] Example 7. The method of Example 6, wherein the composition is delivered at or near a tumor or intratumorally.
[0065] The invention is further demonstrated by way of the figures and data presented herein.
EXAMPLE 1
[0066] This example describes the development of anti-HER2-collagenase fusion proteins. 2Rs15d, a high affinity anti-HER2 sdAb, was employed as a model targeting vector. 2Rs15d binds to an epitope that is distinct from that targeted by trastuzumab and, consequently, 2Rs15d does not compete with trastuzumab or TDM1 for HER2 binding. Clostridium collagenase-H (ColH) was selected as a model matrix-modulating enzyme. 2Rs15d-ColH fusion proteins were expressed in E. coli and characterized for HER2 binding and for collagenase activity.
Methods
Development of 2Rs15d-Collagenase
[0067] DNA encoding for the 2Rs15d-Clostridial Collagenase H fusion protein (2Rs15d-ColH) was synthesized commercially by GenScript (SEQ ID NO:2). The DNA product was digested with XhoI and NdeI restriction enzymes and ligated into the Pet22b(+) plasmid. The E. coli strain SHuffle was transformed with the 2Rs15d-ColH Pet22b vector through heat shock and plated onto a LB agar plate with 100 μg/ml ampicillin and grown overnight at 30° C. Following incubation, a single transformed colony was picked with a sterile pipette tip and inoculated into 5 mL of LB medium and grown overnight at 30° C. in a shaker incubator (200 RPM, 18 hours). Following overnight growth, glycerol stocks were generated through a 1:1 dilution of the transformed SHuffle culture in 50% glycerol and stocks stored at −80° C. To express the 2Rs15d-ColH construct a glycerol stock was removed from the −80° C. and a small volume spread over a LB agar plate (100 μg/ml ampicillin) using a sterile inoculation loop. Following overnight incubation, a single colony was lifted from the LB agar plate and inoculated into a starter culture of LB medium with 100 μg/mL ampicillin in a shaker incubator (30° C., 200 RPM, 18 hours). Following an 18-hour incubation the starter culture was diluted 1/100× into LB medium containing 100 μg/ml ampicillin and grown in a shaker incubator at 30° C., 200 RPM. Cell density was monitored at OD 600 nm using a biospectrophotometer. Once the culture reached an optical density of 0.6-0.8, 2Rs15d-ColH expression was initiated through addition of 1 mM IPTG into the growth medium with expression proceeding for 18-20 hours at 14° C., 200 RPM. Following expression, E. coli cells were pelleted through centrifugation at 10,000×g for 5 minutes. Pelleted cells were lysed using Bugbuster® protein extraction reagent containing 1 mg/ml lysozyme and 0.25 units/mL Benzonase® Nuclease. The cell lysate, containing 2Rs15d-ColH, was passed over a 3 mL HisPur™ Ni-NTA spin column through gravity filtration to allow purification through the C-terminal hexahistidine tag on 2Rs15d-ColH, encoded as part of the Pet22b vector. Non-specifically bound protein was removed from the column using manufacturer recommendations for wash buffer composition and volume. Following washing, 2Rs15d-ColH was eluted using a 500 mM imidazole elution buffer. Following elution, the purified 2Rs15d-ColH was buffer exchanged into PBS using a 5 mL, 7 kDa molecular weight cut-off Zeba™ spin desalting column. The final purified product in PBS was analyzed for purity using SDS-PAGE.
Characterization of 2Rs15d-ColH Enzyme Activity
[0068] A fluorescence-based plate assay was developed to determine the enzyme activity of purified 2Rs15d-ColH. The collagenase substrate fluorescein pig skin gelatin (1 mg per vial) was purchased from ThermoFisher (D12054) and diluted into distilled water at 1 mg/ml. 20 uL of the fluorescein substrate was added to individual wells of a 96 well Nunc MaxiSorp™ ELISA plate and diluted with 80 μl of the recommended activity buffer: 50 mM TRIS, 150 mM NaCl, 5 mM CaCl2 pH 7.6. A standard curve of collagenase activity was generated through serial dilution of a commercially obtained purified clostridium collagenase H (Worthington Biochemical, LS005273) with known activity between 0.05-5 units/mL in activity buffer. 100 uL of each ColH standard was added to the wells of the ELISA plate in duplicate in addition to serial dilutions of the unknown 2Rs15d-ColH and immediately placed into a SpectraMax i3 multi-mode microplate reader. Fluorescence was read every 30 seconds for 5 minutes at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A standard curve of enzyme activity was generated using the observed ΔFluorescence/minute for the ColH standards. The dilution of 2Rs15d-ColH that fell within the linear range of the standard curve was used to determine the number of enzyme units of the purified 2Rs15d-ColH product.
Characterization of 2Rs15d-ColH HER2 Binding Affinity
[0069] Surface plasmon resonance was performed using a Reichert SR7500DC SPR and SR8100 autosampler to assess HER2 binding of the 2Rs15d-ColH product. HER2-Fc (Sino Biological, 10004-H02H) was immobilized onto a carboxymethyl dextran SPR chip (Reichert, 13206066) using EDC/NHS linker chemistry. Purified 2Rs15d-ColH was diluted into the SPR mobile phase (1×PBS, 0.005% Tween-20) at concentrations of 10, 25, 50, 100, 200 and 500 nM. Individual dilutions were injected over the HER2-Fc chip for 1 minute at a flow-rate of 25 μL per minute with a 60-minute dissociation at a flow rate of 25 uL per minute. The chip was regenerated between individual runs through injection of 10 mM Glycine pH 1.5 over the chip surface for 2 minutes at a flow rate of 25 uL per minute. The observed sensor-grams were fit to a 1:1 Langmuir binding model in the Scrubber analysis software to obtain kon, koff, and the equilibrium dissociation constant KD.
Results
[0070] The effects of 2Rs15d-ColH on trastuzumab exposure and TDM1 efficacy in NCI-N87 tumors were evaluated. Initial investigations evaluated the effects of 50 units of untargeted collagenase and 50 units of targeted collagenase. (via the novel fusion protein, 2Rs15d-ColH) on trastuzumab distribution in mice bearing NCI-N87 tumors.
[0071] The production, purification, characterization of anti-HER2-collagenase fusion proteins was carried out. (
[0072] The effects of targeted collagenase fusion protein on trastuzumab efficacy in mice bearing HER2+ tumors is shown in
EXAMPLE 2
[0073] This example describes a fusion protein comprising ColH with an N-terminal anti-HER2 single domain antibody and a C-terminal albumin binding domain.
Materials and Methods
[0074] Antibody/Cell-Lines/Mouse Models
[0075] Trastuzumab and T-DM1 were purchased from Millard Fillmore Memorial Hospital (Amherst, N.Y.). The gastric carcinoma cell-line NCI-N87 was cultured following American Type Culture Collection recommendations. Male Nu/J mice were purchased from The Jackson Laboratory (Bar Harbor, Me.), and male Swiss-Webster mice were purchased from Envigo (Indianapolis, Ind.). Nu/J mice were injected subcutaneously in the right flank with 100 μL of a 1:1 solution of matrigel (Thermo Fisher Scientific, Waltham, Mass., CB-40234):RPMI 1640 containing 5 million NCI-N87 cells for the T-DM1 efficacy study and with 200 μl of a 1:1 matrigel:RPMI 1640 solution containing 5 million NCI-N87 cells for the fluorescence studies.
Fusion Protein Sequences
[0076] Examples of configurations for fusion construct comprising 2Rs15d, 2Rb17c, ColH and ABD035 are shown in
Expression and Purification of Collagenase Fusion Proteins
[0077] DNA encoding for the fusion protein was digested with XhoI and NdeI restriction enzymes and ligated into the Pet22b(+) plasmid (Millipore-Sigma, Burlington, Mass., 69744). The E. coli strain SHuffle® (NEB, C3029J, Ipswich, Mass.) was transformed with the fusion protein DNA ligated into the Pet22b vector through heat shock and plated onto a lysogeny broth (LB) agar plate with 100 μg/ml ampicillin and grown overnight at 30 degrees Celsius (° C.). Following incubation, a single transformed colony was picked with a sterile pipette tip and inoculated into 5 mL of LB medium and grown overnight at 30° C. in a shaker incubator set at 200 rotations per minute (RPM) for 18 hours. Following overnight growth, glycerol stocks were generated through a 1:1 dilution of the transformed SHuffle® culture in 50% glycerol and stocks stored at −80° C. Fusion proteins were expressed by removal of a glycerol stock from −80° C. storage, and a small volume spread over an LB agar plate (100 μg/ml ampicillin) using a sterile inoculation loop. Following overnight incubation, a single colony was lifted from the LB agar plate and inoculated into a starter culture of LB medium with 100 μg/mL ampicillin in a shaker incubator (30° C., 200 RPM, 18 hours). Following an 18-hour incubation, the starter culture was diluted 1/100 into LB medium containing 100 μg/ml ampicillin and grown in a shaker incubator at 30° C., 200 RPM. Cell density was monitored at a wavelength of 600 nanometers (nm) using a spectrophotometer. Once the culture reached an optical density of 0.6-0.8, protein expression was initiated through the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) into the growth medium with expression proceeding for 18-20 hours at 12° C., 200 RPM. Following expression, SHuffle® cells were pelleted through centrifugation at 10,000 relative centrifugal force (RCF) for 5 minutes. Pelleted cells were lysed using Bugbuster® (Millipore-Sigma, Burlington, Mass., 70584) protein extraction reagent containing 1 mg/ml lysozyme and 0.25 units/mL Benzonase® Nuclease (Millipore-Sigma, Burlington, Mass., 70584). The cell lysate, containing fusion protein, was passed over a 3 mL HisPur™ Ni-NTA spin column (Thermo Fisher Scientific, Waltham, Mass., 88226) through gravity filtration to allow purification through the C-terminal hexahistidine tag, encoded as part of the Pet22b vector. Non-specifically bound protein was removed from the column using manufacturer recommendations for wash buffer composition and volume. Following washing, the fusion protein was eluted using a 500 mM imidazole elution buffer. Following elution, the fusion protein was buffer exchanged into phosphate buffered saline buffer pH 7.4 (PBS) using a 5 mL, 7 kDa molecular weight cut-off Zeba™ spin desalting column (Thermo Fisher Scientific, Waltham, Mass., 89891). The final purified product in PBS was evaluated using sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE).
Characterization of 2Rs15d-ColH Enzyme Activity
[0078] A fluorescence-based plate assay was developed to determine the enzyme activity of purified fusion protein. The collagenase substrate fluorescein pig skin gelatin (1 mg per vial) was purchased from Thermo Fisher Scientific (Waltham, Mass., D12054) and diluted into distilled water (dH2O) at 1 mg/ml. 20 microliters (μL) of the fluorescein substrate was added to individual wells of a 96 well Nunc MaxiSorp™ plate (Thermo Fischer Scientific, Waltham, Mass., 439454) and diluted with 80 μL of a buffer containing 50 mM tris(hydroxyethyl)aminomethane, 150 mM sodium chloride, 5 mM calcium chloride pH 7.6. A standard curve of collagenase activity was generated through serial dilution of a commercially obtained ColH (Worthington Biochemical, Lakewood, N.J., LS005273) with known activity between 0.05-5 units/milliliter (U/mL) in activity buffer. 100 μL of each ColH standard was run in duplicate with duplicate samples of 10×, 100× and 1000× dilutions of the fusion protein and immediately placed into a SpectraMax i3 multi-mode microplate reader (Molecular Devices, San Jose, Calif.). The fluorescence was read every 30 seconds for 5 minutes at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A standard curve of enzyme activity was generated using the observed change in fluorescence/minute for the ColH standards. The fusion protein dilution that fell within the linear range of the standard curve was used to determine the number of enzyme units of the purified product.
Surface Plasmon Resonance
[0079] Surface plasmon resonance (SPR) was performed using a Reichert SR7500DC SPR and SR8100 autosampler (Reichert, Depew, N.Y.). HER2-Fc (Sino Biological, Beijing, China, 10004-H02H) or mouse serum albumin (MSA) (MyBioSource, San Diego, Calif., MBS135633) was immobilized onto a carboxymethyl dextran SPR chip (Reichert, Depew, N.Y., 13206066) using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide linker chemistry. A mobile phase of PBS 0.05% Tween-20 at a flow rate of 25 μL/min was used for all binding evaluations. Fusion protein dilutions were injected for 90 seconds, and the chip regenerated following dissociation with a 2-minute injection of a 10 millimolar (mM) glycine buffer pH 1.5. HER2 binding kinetics for 2Rs15d-ColH-ABD was evaluated at concentrations of 25, 50, 100, 200 and 500 nanomolar (nM) with a 60-minute dissociation time. HER2 binding kinetics for 2Rb17c-ColH-ABD was assessed at concentrations 10, 25, 50, 100, 250 nM with a 5-minute dissociation. MSA binding for 2Rs15d-ColH-ABD was evaluated at concentrations of 7.5, 15, 30 and 60 nM with a 10-minute dissociation time. MSA binding for 2Rb17c-ColH-ABD was assessed at concentrations of 7.5, 37.5 and 75 nM with a 10-minute dissociation time. The observed sensor-grams were fit to a 1:1 Langmuir binding model in the Scrubber analysis software to obtain the association rate constant (kon), dissociation rate constant (koff), and the equilibrium dissociation constant (K.sub.D).
Plasma Pharmacokinetics of 2Rs15d-ColH and 2Rs15d-ColH-ABD
[0080] Male Swiss-Webster mice were intravenously injected through the penile vein with 2Rs15d-ColH-ABD and 2Rs15d-ColH at a dose of 2000 collagenase units/kilogram bodyweight (U/kg) (3 mice/group). Blood samples were collected at 5, 15, 30, and 60 minutes after injection with lithium heparin as the anti-coagulant. Blood samples were centrifuged for 5 minutes at 500 RCF and plasma collected by pipetting. Plasma samples were analyzed for collagenase activity using the fluorescence collagen assay described above with the following changes. The activity of the 5-minute timepoint for 2Rs15d-ColH-ABD and 2Rs15d-ColH was determined using a 60-minute incubation with a standard curve of enzyme activity between 1-10 U/mL. The enzyme activity of the 15-minute time point for 2Rs15d-ColH-ABD and 2Rs15d-ColH and the 30-minute time point for 2Rs15d-ColH-ABD was determined using a 3-hour incubation with ColH standards between 0.25 and 2.5 U/mL. The 30-minute time point for the 2Rs15d-ColH and the 60-minute time point for 2Rs15d-ColH-ABD and 2Rs15d-ColH was analyzed following an 18-hour incubation with a standard curve between 0.025 and 0.5 U/mL. Area under the curve (AUC) values between 5 and 60 minutes were calculated for individual mice using the linear trapezoidal method.
Fluorescent Assessment of Trastuzumab Distribution
[0081] Trastuzumab was labeled with Alexa-Fluor 680 (Thermo Fisher Scientific, Waltham, Mass., A20188) following manufacturer recommendations with the recommended antibody concentration, and the volume doubled to limit over modification. NU/J mice bearing NCI-N87 xenograft tumors were administered 2 mg/kg of Alexa-Fluor680 labeled trastuzumab through retro-orbital injection. Ten minutes after trastuzumab administration, 2000 U/kg 2Rs15d-ColH-ABD035 was administered via penile vein injection with control mice administered a volume equivalent of PBS (2 mice/group). Mice were sacrificed 24 hours post-injection, tumors resected, covered in OCT freezing media (VWR, Radnor, Pa., 25608-930) and frozen in isopentane cooled liquid nitrogen. Frozen tumors were sectioned using an HM525 cryostat (Microm, Walldorf, Germany) at a slice thickness of 10 μM. Tumor sections were outlined with a PAP pen (Newcomersupply, Middleton, Wis., 6505) and covered with 1% mouse plasma in PBS buffer with a 1:50 dilution of a rat anti-mouse CD31 antibody (Invitrogen, Carlsbad, Calif., 390) labeled with Alexa-Flour555 (Thermo Fisher Scientific, Waltham, Mass., A20187). Tumors sections were stained for 30 minutes, followed by three five-minute PBS washes. Tumor sections were mounted in FluorSave (Millipore-Sigma, Burlington, Mass., 345789) and imaged identically using an EVOS Fl autofluorescent microscope (Thermo Fisher Scientific, Waltham, Mass.) with red fluorescent protein and cyanine 5 excitation cubes. Three slices were imaged per tumor and the mean fluorescence and percent area above threshold determined in ImageJ (NIH, Rockville, Md.). The mean of three slices was used to represent an individual tumor and statistical significance between the two groups determined in GraphPad Prism 7 (GraphPad, San Diego, Calif.) using Student's t-test. Selected tumor images that are shown in
Fluorescent Assessment of Tumor Collagen
[0082] Male NU/J mice bearing NCI-N87 xenograft tumors were administered either PBS (n=1) or 2000 U/kg 2Rs15d-ColH-ABD035 (n=1) by penile vein injection. 8 hours after injection, mice were sacrificed, and tumors resected, frozen, and cryo-sectioned following the above protocol. Tumor collagen and vasculature were immunofluorescently stained for 1-hour using a 1:100 dilution of an Alexa-Flour680 conjugated anti-collagen antibody (Invitrogen, Carlsbad, Calif., PA5-29569) and a 1:50 dilution of a rat anti-mouse CD31 antibody (Invitrogen, Carlsbad, Calif., 390) labeled with Alexa-Flour555 (Thermo Fisher Scientific, Waltham, Mass., A20187). Tumors were washed, mounted and imaged following the above protocol. For image clarity, the images shown in
NCI-N87 Efficacy
[0083] Mice bearing NCI-N87 xenografts at a volume of 250 mm.sup.3 were split into six treatment groups of (i) PBS vehicle (ii) 2000 U/kg 2Rs15d-ColH (iii) 1.8 mg/kg T-DM1 (iv) 1.8 mg/kg T-DM1 and 2000 U/kg ColH, (v) 1.8 mg/kg T-DM1 and 2000 U/kg 2Rs15d-ColH and (vi) 1.8 mg/kg T-DM1 and 2000 U/kg 2Rs15d-ColH-ABD. All groups had a total of six mice except for group iii, which had 7 mice. T-DM1 or PBS was administered via retro-orbital injection, and collagenase/PBS administered 10 minutes after the T-DM1/PBS administration via penile vein injection. Mice were observed for any signs of distress or bruising at the site of injection. Tumors were measured with digital Vernier calipers and volumes calculated using the formula L.sup.2×W/2, were L is the longest diameter of the tumor and W the shortest. Upon reaching a tumor volume of 1200 mm.sup.3 mice were sacrificed. Tumor volumes for individual mice, up to 14 days after injection, were fit to a mono-exponential growth function in GraphPad Prism 7. Best fit growth function (Kgex) values were compared between the groups with Student's t-test and Bonferroni's correction for multiple comparisons.
Results
Fusion Protein Expression and Functional Activity
[0084] 2Rs15d- and 2Rb17c-ColH-ABD were successfully expressed in E. Coli. SDS-PAGE gels for the expression and purification for both constructs are shown in
2Rs15d-ColH-ABD and 2Rs15d-ColH Plasma Pharmacokinetics
[0085] 2Rs15d-ColH and 2Rs15d-ColH-ABD were administered to Swiss-Webster mice at a dose of 2000 U/kg and plasma activity determined at 5-, 15-, 30- and 60-minutes following administration. Plasma time profiles for both constructs are shown in
[0086] Impact of 2Rs15d-ColH-ABD on Trastuzumab Uptake and Tumor Collagen Content
[0087] Co-administration of 2Rs15d-ColH-ABD dramatically increased the uptake of AF680-trastuzumab in NCI-N87 xenografts in comparison to AF680-trastuzumab administered alone (
Impact of ColH on T-DM1 efficacy in NCI-N87 Xenografts
[0088] T-DM1 led to a significant increase in group survival in comparison to PBS or 2Rs15d-ColH alone (p<0.05). Likely resulting from the small group size and high intra-group variability, a significant extension in survival was not observed between the T-DM1 groups (
[0089] While the invention has been described through illustrative examples, routine modifications will be apparent to those skilled in the art, which modifications are intended to be within the scope of the invention.