WO2005071073A1

WO2005071073A1 - Fap compositions and the use thereof for immunomodulation

Info

Publication number: WO2005071073A1
Application number: PCT/US2005/000709
Authority: WO
Inventors: Paul A. Mclean; Barry Jones; Glenn T. Miller; Michael I. Jesson
Original assignee: Point Therapeutics, Inc.
Priority date: 2004-01-09
Filing date: 2005-01-10
Publication date: 2005-08-04

Abstract

The invention provides compositions and methods for down-regulating immune responses using FAP alpha dimer enzyme.

Description

FAP COMPOSITIONS AND THE USE THEREOF FOR IMMUNOMODU ATION

Field of the Invention The invention relates to compositions of fibroblast activation protein (FAP) alpha dimer enzyme for use in immunomodulation, and more particularly immunoinhibition.

Background of the Invention Dipeptidyl peptidase activity is characterized by the cleavage of dipeptides from the ends of polypeptides. CD26/dipeptidyl peptidase IN (DPPIN) and fibroblast activation protein alpha (FAP alpha) are integral membrane proteins present on the cell surface of certain mammalian cell types.^28"31 Both proteins are enzymes that cleave amino-terminal dipeptides where the penultimate amino acid is either proline or alanine. CD26 DPPIN has been shown to digest biologically active polypeptides such as chemokines and short polypeptide hormones in experimental systems.³² The biologically relevant targets of FAP have hitherto not been identified. . There exist structural similarities between the catalytic sites of CD26/DPP-1N and FAP as well as overlapping substrate specificities. However, notwithstanding the shared specificity for polypeptides with amino-terminal dipeptides ofthe sequence X-Pro (Ala), where X can be any natural L-amino acid, FAP was initially distinguished from CD26/DPPIN by its ability to completely digest gelatin³⁵. The pattern of tissue expression of FAP also differs from CD26/DPPIN. FAP alpha is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. Normal adult tissues are generally FAP alpha-negative, but some fetal mesenchymal tissues transiently express the molecule. FAP demonstrates a restricted normal tissue distribution and abundant expression in the stroma of over 90% of breast, colorectal, and lung carcinomas. In contrast, CD26/DPPIN expression is widespread in healthy tissues. For example, it can readily be detected in epithelial and lymphoid tissue. In the latter, thymus-derived lymphocytes (T cells) have been shown to express the protein in greater amounts when the cells become activated by antigenic stimulation ' . Whether FAP or CD26 is involved in regulating a particular biologically active polypeptide by Ν-terminal degradation might be determined by difference in anatomical expression or subtle differences in substrate specificity between the two enzymes: such as preference for certain N-terminal residues preceding Pro/ Ala , polypeptide size or post-translational modification .

Summary of the Invention The invention relates in part to methods for increasing FAP alpha dimer enzyme activity and compositions of FAP alpha dimer enzyme. In accordance with the invention, increases in FAP alpha dimer enzyme activity levels have therapeutic benefit, for example, in immunomodulation, and more particularly immunoinhibition. Some methods provided herein harness the natural ability of FAP alpha dimer enzyme to modulate IL-1 production in vivo. The invention further provides compositions of FAP alpha dimer enzyme that can be used, inter alia, in the methods ofthe invention. Thus, in one aspect, the invention provides a method for down-regulating an immune response comprising administering to a subject in need thereof a FAP alpha dimer enzyme in an amount effective to down-regulate an immune response. In one embodiment, the immune response is an IL-1 mediated condition. In another embodiment, the immune response is an abnormal immune response such as but not limited to inflammation, autoimmune disease, sepsis, graft versus host disease, transplant rejection, toxic shock syndrome, allergy, asthma, atherosclerosis, osteoarthritis, and Guillain-Barre's syndrome. In another embodiment, the abnormal immune response is subsequent to an infection, such as but not limited to an RSN infection. The autoimmune disease may be selected from the group consisting of rheumatoid arthritis, insulin dependent diabetes (type I diabetes), inflammatory bowel disease, autoimmune thyroiditis, systemic lupus erythematosus (SLE), uveitis, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre's syndrome, psoriasis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis and multiple sclerosis. In one embodiment, the subject does not have cancer and/or the subject does not have a predisposition to cancer. In one embodiment, the method further comprises administering to the subject a second agent. The second agent may be an anti-inflammatory agent, an immunosuppressant, or an anti-infective agent, but it is not so limited. The anti-infective agent may be an antibacterial agent, an anti- viral agent, an anti-fungal agent, an anti-parasitic agent, or an anti- mycobacterial agent. In another embodiment, the FAP alpha dimer enzyme is wild type FAP alpha dimer enzyme. The FAP alpha dimer enzyme may be a truncation mutant or a fusion or chimeric protein. The fusion or chimeric protein may comprise a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a self-cleaving affinity tag, a tissue localization sequence and a peptide or protein ligand. Examples of secretion sequences include but are not limited to a G-CSF leader sequence or an Ig-kappa leader sequence. Examples of purification sequences include but are not limited to GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain. Examples of epitopes include but are not limited to a hemaglutimiin tag, a FLAG tag, a N5 tag, a myc tag and a T7 tag. The protein degradation sequence may be a PEST sequence but it is not so limited. Examples of protease cleavage site include but are not limited to enterokinase, factor Xa protease, thrombin, TEN protease, PreScission protease, Furin, and Genenase. Unless otherwise indicated, the point mutations recited herein correspond to the amino acid of human FAP, as indicated in SEQ ID NO: 2. In one embodiment, the fusion or chimeric protein comprises an amino acid substitution of Q732E or N733D. In another embodiment, the FAP alpha dimer enzyme is a heterodimer. The heterodimer may be a heterodimer of a FAP alpha monomer and a DPPIN/CD26 monomer, but it is not so limited. In another embodiment, the FAP alpha dimer enzyme comprises an amino acid substitution (as compared to or relative to wild type FAP alpha dimer amino acid sequence). The amino acid substitution may be present in the β-propeller domain. The amino acid substitution may be at positions Y124, A207, A347, G349, F351 or N352. Specific examples of amino acid substitutions include but are not limited to Y124H, A207S, A347N, G349R, F351R and N352P. In another embodiment, the amino acid substitution is present in the catalytic domain. As an example, the amino acid substitution may be in amino acid A657, such as A657D. The amino acid substitution may be Y124H or A207S. Other examples of amino acid substitutions include but are not limited to A347N, G349R, F351R or N352P. In still another embodiment, the amino acid substitution is present in the entrance to the catalytic site. In a related embodiment, the entrance to the catalytic site is an apical entrance. The amino acid substitution may be selected from the group consisting of G64D, Q65H, V299A, D301Q, T354E, N356H, S358T, Y359L, F401E, R402A, N403L, Q405S, T452S, A453N, D457K and Y458E. In another embodiment, the entrance is a side entrance, and optionally the amino acid substitution may be selected from the group consisting of Ν49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, N98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q and Y124H. In yet another embodiment, the amino acid substitution is present at an N-linked glycosylation site. Examples of N-linked glycosylation site is selected from the group consisting of N49, N92, N99, N227, N314 and N679. The amino acid substitution may also be at T51, T94, SI 01, T229, S316 or T681. In particular embodiments, the amino acid substitution is at N227 and T229. In still another embodiment, the amino acid substitution is T229M. In any ofthe foregoing embodiments, the FAP alpha dimer enzyme may also be soluble and have the recited mutations overlayed thereon. In another embodiment, the amino acid substitution alters disulfide bond formation. For example, the amino acid substitution may introduce a disulfide bond. In another embodiment, the amino acid substitution is selected from the group consisting of H378C and A386C. The amino acid substitution may be selected from the group consisting of L48C, N742C, M683C and 1713C. In another related embodiment, the amino acid substitution removes a disulfide bond. In one embodiment, the FAP alpha dimer enzyme is PEGylated. In a related embodiment, the FAP alpha dimer enzyme is PEGylated at a lysine or at a cysteine. For example, the FAP alpha dimer enzyme may be PEGylated at a cysteine introduced at position 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, 753, 91, 148, 263, 323, 343, or 444 (relative to wild type sequence). In another embodiment, the PEGylated FAP alpha dimer enzyme comprises a mutation in one or more amino acid positions selected from a group consisting of 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, 753, 91, 148, 263, 323, 343 and 444. In another embodiment, the FAP alpha dimer enzyme is a dimerization domain mutant. In a related embodiment, the dimerization domain mutant lacks residues comprised of P232-I250 of wild type FAP alpha dimer enzyme and comprises residues P234-N254 of wild type DPPIN. In another embodiment, the dimerization domain mutant lacks residues F706-D731 of wild type FAP alpha dimer enzyme or some portion thereof and comprises residues F713-D738 of wild type DPPIV or some portion thereof. In still another embodiment, the dimerization domain mutant comprises an amino acid substitution of T248C. In another embodiment, the FAP alpha dimer enzyme lacks residues Ν679-Ν733 from wild type FAP alpha dimer enzyme and comprises residues N685-D739 of wild type DPPIN. In yet another embodiment, the amino acid substitution is present in the cytoplasmic domain. The FAP alpha dimer enzyme may lack a cytoplasmic domain. In still a further embodiment, the amino acid substitution is present in the transmembrane domain. The FAP alpha dimer enzyme may lack a transmembrane domain. The FAP alpha dimer enzyme may lack a cytoplasmic and transmembrane domain. For example, the FAP alpha dimer enzyme lacks residues corresponding to 1-37 from wild type FAP alpha dimer enzyme. In preferred embodiments, the FAP alpha dimer enzyme is soluble. In some embodiments, the FAP alpha dimer enzyme comprises an amino acid substitution of T229M. In other embodiments, it does not. In some embodiments, the FAP alpha dimer enzyme comprises an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 61, which may optionally be overlayed with one or more ofthe mutations discussed herein. The FAP alpha dimer enzyme may be administered as a protein or as a nucleic acid. In another embodiment, IL-1 is IL-1 alpha or IL-1 beta. In another aspect, the invention provides a pharmaceutical preparation comprising a

FAP alpha dimer enzyme in a pharmaceutically acceptable carrier, wherein the preparation is sterile and lacks an adjuvant. In another aspect, the invention provides a pharmaceutical preparation comprising a FAP alpha dimer enzyme in a pharmaceutically acceptable carrier, and a non-adjuvant second agent. In one embodiment, the non-adjuvant second agent is an anti-inflammatory agent or an immunosuppressant. In another embodiment, the preparation is sterile. In various embodiments, the FAP alpha dimer enzyme is wild type FAP alpha dimer enzyme. The FAP alpha dimer enzyme may also be a truncation mutant. In yet another embodiment, the FAP alpha dimer enzyme is a fusion or chimeric protein. The fusion or chimeric protein may comprise a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a tissue localization sequence, a peptide or protein ligand. Examples of secretion sequences include but are not limited to a G-CSF leader sequence or an Ig-kappa leader sequence. Examples of purification sequences include but are not limited to a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain. Examples of epitopes include but are not limited to a hemaglutinnin tag, a FLAG tag, a V5 tag, a myc tag and a T7 tag. An example of a protein degradation sequence is a PEST sequence. Examples of protease cleavage sites include but are not limited to sites recognized by enterokinase, factor Xa protease, thrombin, TEN protease, PreScission protease, Furin, Genenase. In one embodiment, the fusion or chimeric protein comprises an amino acid substitution of Q732E or Ν733D. In another embodiment, wherein the FAP alpha dimer enzyme is a heterodimer. The heterodimer may be a heterodimer of a FAP alpha monomer and a DPPIN/CD26 monomer. In another embodiment, the FAP alpha dimer enzyme comprises an amino acid substitution (as compared to or relative to wild type FAP alpha dimer). In one embodiment, the amino acid substitution is present in the β-propeller domain.

In a related embodiment, the substitution is at Y124, A207, A347, G349, F351, N352, and can include but is not limited to Y124H, A207S, A347N, G349R, F351R, V352P. In another embodiment, the amino acid substitution is present in the catalytic domain. In a related embodiment, the amino acid substitution is selected from the group consisting of Y124H, A207S, A347N, G349R, F351R, V352P and A657D. In still another embodiment, the amino acid substitution is at A657. In a related embodiment, the amino acid substitution is A657D. The amino acid substitution may be Y124H or A207S. The amino acid substitution may also be A347N, G349R, F351R or N352P. In another embodiment, the amino acid substitution is present in the entrance to the catalytic domain. The entrance to the catalytic domain may be an apical entrance. The amino acid substitution may be selected from the group consisting of G64D, Q65H, N299A, D301Q, T354E, N356H, S358T, Y359L, F401E, R402A, N403L, Q405S, T452S, A453N, D457K and Y458E. The entrance to the catalytic domain may be a side entrance. The amino acid substitution may be selected from the group consisting of Ν49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y. D117N, S119N, L121Q and Y124H. In another embodiment, the amino acid substitution may be present at an Ν-linked glycosylation site. The Ν-linked glycosylation site may be selected from the group consisting of Ν49, N92, N99, N227, N314 and N679. In one embodiment, the amino acid substitution is T229M. In some preferred embodiments, the FAP alpha dimer enzyme is soluble. In still another embodiment, the amino acid substitution alters disulfide bond formation. For example, the amino acid substitution may introduce a disulfide bond. In a related embodiment, the amino acid substitution is selected from the group consisting of H378C and A386C. In another related embodiment, the amino acid substitution is selected from the group consisting of L48C, N742C, M683C and I713C. As another example, the amino acid substitution removes a disulfide bond. In another embodiment, FAP alpha dimer enzyme is PEGylated. In yet another embodiment, FAP alpha dimer enzyme is a dimerization domain mutant. In a related embodiment, the dimerization domain mutant lacks residues P232-I250 of wild type FAP alpha dimer enzyme and comprises residues P234-N254 of wild type

DPPIN. In another embodiment, the dimerization domain mutant lacks residues F706-D731 of wild type FAP alpha dimer enzyme and comprises residues F713-D738 of wild type DPPIN. In yet another embodiment, dimerization domain mutant comprises an amino acid substitution of T248C. In yet another embodiment, the FAP alpha dimer enzyme lacks residues Ν679-Ν733 from wild type FAP alpha dimer enzyme and comprises residues N685- D739 ofwild type DPPIN. In still another embodiment, the amino acid substitution is present in the cytoplasmic domain. The amino acid substitution may be present in the transmembrane domain. In one embodiment, the FAP alpha dimer enzyme lacks a cytoplasmic domain and/or a transmembrane domain. In a related embodiment, the FAP alpha dimer enzyme lacks residues corresponding to 1-37 from wild type FAP alpha dimer enzyme (SEQ ID NO: 70). The FAP alpha dimer enzyme may comprise an amino acid substitution of T229M. In various embodiments, the FAP alpha dimer enzyme comprises an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 61 or SEQ ID NO:70, optionally overlayed with one or more ofthe amino acid substitutions or other mutations recited herein. In still another embodiment, the FAP alpha dimer enzyme is present in an amount effective to down-regulate an immune response. In still another aspect, the invention provides a composition comprising a FAP alpha dimer enzyme comprising an amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 70, and optionally (1) one or more amino acid substitutions selected from the group consisting of Y124H, A207S, A347N, G349R, F351R, N352P, A657D, Q732E, Ν733D, G64D, Q65H, N299A, D301Q, T354E, N356H, S358T, Y359L, F401E, R402A, N403L, Q405S, T452S, A453N, D457K, Y458E, Ν49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q, Y124H, H378C, A386C, L48C, N742C, M683C, I713C and T248C, (2) lacking residues P232-I250 and comprising residues P234-N254 of wild type DPPIN, (3) lacking residues F706-D731 and comprising residues F713-D738 of wild type DPPIN, (4) lacking residues Ν679-Ν733 and comprising residues N685-D739 of wild type DPPIN, or (5) an amino acid substitution of T229M. In one embodiment, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 Ν-terminal amino acids in SEQ ID NO: 61 are deleted. In one embodiment, the FAP alpha dimer enzyme is a fusion or chimeric protein. The fusion or chimeric protein may comprise a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a self-cleaving affinity tag, a tissue localization sequence and a peptide or protein ligand. The secretion sequence may be a G-CSF leader sequence or an Ig-kappa leader sequence. The purification sequence may be selected from the group consisting of a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain. The epitope may be selected from the group consisting of a hemaglutimiin tag, a FLAG tag, a N5 tag, a myc tag and a T7 tag. The protein degradation sequence may be a PEST sequence. The protease cleavage site may be selected from the group consisting of enterokinase, factor Xa protease, thrombin, TEN protease, PreScission protease, Furin, and Genenase. In one embodiment, the FAP alpha dimer enzyme is a heterodimer. The heterodimer may be a heterodimer of a FAP alpha monomer and a DPPIN/CD26 monomer. In another embodiment, the amino acid substitution is A657D. In yet another embodiment, the amino acid substitution is Y124H or A207S. In still another embodiment, the amino acid substitution is A347N, G349R, F35 IR or

N352P. In yet a further embodiment, the amino acid substitution is selected from the group consisting of G64D, Q65H, N299A, D301Q, T354E, N356H, S358T, Y359L, F401E, R402A, N403L, Q405S, T452S, A453N, D457K and Y458E or from the group consisting of Ν49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q and Y124H. In important embodiments, the FAP alpha dimer enzyme is soluble. The FAP alpha dimer enzyme may lack residues P232-1250 and may comprise residues P234-V254 of wild type DPPIV. In one embodiment, the dimerization domain mutant lacks residues F706-D731 and comprises residues F713-D738 of wild type DPPIV. In another embodiment, the FAP alpha dimer enzyme lacks residues N679-N733 and comprises residues N685-D739 of "wild type DPPIV. In yet another aspect, the invention provides a composition comprising a FAP alpha dimer enzyme comprising an amino acid substitution of A657D. In one embodiment, the FAP alpha dimer enzyme is soluble. In another embodiment, the FAP alpha dimer enzyme further comprises an amino acid substitution of T229M. In yet another embodiment, the FAP alpha dimer enzyme further comprises an amino acid substitution of Y124H or A207S. In still another embodiment, the FAP alpha dimer enzyme further comprises an amino acid substitution of A347V, G349R, F351R or V352P. In one embodiment, the FAP alpha dimer enzyme is a fusion or chimeric protein. Various embodiments of fusion or chimeric proteins have been recited above and apply to this aspect ofthe invention. In another embodiment, the heterodimer is a heterodimer of a FAP alpha monomer and a DPPIV/CD26 monomer. In yet another embodiment, the amino acid substitution is A347V, G349R, F351R or V352P. The FAP alpha dimer enzyme may further comprise an amino acid substitution of G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K or Y458E or an amino acid substitution of N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q or Y124H. In one embodiment, the FAP alpha dimer enzyme lacks residues P232-I250 and comprises residues P234-V254 of wild type DPPIV. In another embodiment, the dimerization domain mutant lacks residues F706-D731 and comprises residues F713-D738 of wild type DPPIV. In still another embodiment, the FAP alpha dimer enzyme lacks residues N679-N733 and comprises residues N685-D739 of wild type DPPIV. In a further aspect, the invention provides a composition comprising a FAP alpha dimer enzyme lacking amino acids 269-448 and comprising amino acids 269-448 from mouse FAP. These and other objects ofthe invention will be described in further detail in connection with the detailed description ofthe invention.

Brief Description of the Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of human wild type FAP alpha dimer (GenBank Accession Number NM_004460). SEQ ID NO: 2 is the amino acid sequence of human wild type FAP alpha dimer

(GenBank Accession Number NM_004460). SEQ ID NO: 3 is the nucleotide sequence of a human soluble FAP alpha dimer enzyme as contained in plasmid #122. SEQ ID NO: 4 is the amino acid sequence of a human soluble FAP alpha dimer enzyme as coded in plasmid #122 (having vector derived DAAQPA at the N-terminus, a "TKRA" at the FAP derived N-terminal sequence due to the primer used, .and a T229M mutation). SEQ ID NO: 5 is the nucleotide sequence of wild type murine FAP alpha dimer enzyme (GenBank Accession Number Y10007). SEQ ID NO: 6 is the amino acid sequence of wild type murine FAP alpha dimer enzyme (GenBank Accession Number Y10007). SEQ ID NO: 7 is the amino acid sequence of hDPPIV dimerization region 1 loop. SEQ ID NO: 8 is the amino acid sequence of hFAP alpha dimer enzyme dimerization region 1 loop. SEQ ID NO: 9 is the amino acid sequence of hDPPIV dimerization region 2 loop. SEQ ID NO: 10 the amino acid sequence of hFAP alpha dimer enzyme dimerization region 2 loop. SEQ ID NO: 11 the nucleotide sequence of PCR primer hFAPl (CCACGCTCTG AAGACAGAAT TAGC). SEQ ID NO: 12 is the nucleotide sequence of PCR primer hFAP2 (TCAGATTCTG ATAGAGGCTTGC). SEQ ID NO : 13 is the nucleotide sequence of PCR primer Sfi-FAP-B

(GTAGTCGGCC CAGCCGGCCA CAAAGAGAGC TCTTACCCTG AAGGATATTT TAAATG). SEQ ID NO: 14 is the amino acid sequence ofthe N-terminal six amino acids in mature soluble FAP alpha dimer enzyme derived from the vector pSecTag2-B (DAAQPA). SEQ ID NO: 15 is the amino acid sequence ofthe N-terminal residues in FAP alpha dimer enzyme derived from the vector pSecTag2-B (i.e., excludes 6 vector-derived amino acids shown as SEQ ID NO: 14, and having a "TKRA" at the FAP derived N-terminus due to the primer used) (TKRALTLKDILNG). SEQ ID NO: 16 is the amino acid sequence ofthe first 51 amino acids of wild-type hDPPIV N-terminus excerpted from GenBank Accession NM_010074: (MKTPWKVLLG LLGAAALVTI ITVPVVLLNK GTDDATADSR KTYTLTDYLKN). SEQ ID NO: 17 is the amino acid sequence of serum DPPIV N-terminal sequence #1 (SRKTYTLTDYLKN). SEQ ID NO: 18 is the serum DPPIV N-terminal sequence #2 (RKTYTLTDYLKN). SEQ ID NO: 19 is the first 50 amino acids of wild-type hFAP N-terminus excerpted from

GenBank Accession NM_004460 (SEQ ID NO: 2) (MKTWVKIVFG NATSAVLALL NMCINLRPSR NHΝSEEΝ TMRALTLKDILΝG) SEQ ID NO: 20 is the amino acid sequence ofthe proposed soluble hFAP alpha dimer enzyme N-terminus without the 6 amino acids imparted by the vector (TMRALTLKDILNG). SEQ ID NO: 21 is amino acid sequence ofthe first 50 amino acids of wild-type mouse FAP alpha dimer enzyme N-terminus excerpted from GenBank Accession Y 10007 (excerpted from SEQ ID NO: 6) (MKTWLKTVFG VTTLAALALV VICIVLRPSR VYKPEGN TKRALTLKDILNG). • SEQ ID NO: 22 is the amino acid sequence ofthe proposed soluble mFAP alpha dimer enzyme N-terminus without the 6 amino acids imparted by the vector (TKRALTLKDILNG). SEQ ID NO: 23 is the nucleotide sequence upstream ofthe Sfil site of pSecTag2 vector (InVitrogen) showing the published signal cleavage site is between the 11^th and 12^th codons as follows:

(GTACTG CTG CTCTGG GTT CCAGGTTCCACT GGTGAC GCG GCC CAG CCG GCC). SEQ ID NO: 24 is the amino acid sequence in the region ofthe signal cleavage site and Sfil site in the vector pSecTag2 (VLLLWVPGSTGDAAQPA). SEQ ID NO: 25 is the amino acid sequence encoded by Sfi-FAP-B primer and having the murine "TKRA" at the FAP N-terminus (g DAAQPATKRA LTLKDILNG). SEQ ID NO: 26 is nucleotide sequence ofthe PCR primer hG-CSF F primer (CCAAGCTG GCTAGC CACCATG GCTGGAC CTGCCACCCAGAG). SEQ ID NO: 27 is nucleotide sequence ofthe hG-CSF leader-R primer (GGC TTC CTG CAC TGT CCA GAG TGC ACT). SEQ ID NO: 28 is nucleotide sequence of the hG-CSF_FAP-F primer

(GCACTCTGGA CAGTGCAGGA AGCC ACAAAG AGAGCTCTTA CCcTGAAGGA TATTTTA). SEQ ID NO: 29 is the nucleotide sequence ofthe Xbal site such as hFAP-Clal-R (GCA GGG TAA GTG GTA TCG ATA ATA AAT ATC CG). SEQ ID NO: 30 is the nucleotide sequence ofthe PCR primer Sfi-DPPIV

(GTAGTCGGCC CAGCCGGCCAGTCGCAAAACTTACACTCTAACTGATTAC TTAAAAAAT). SEQ ID NO: 31 is the nucleotide sequence ofthe PCR primer DPP4-R (GTCGGAGCGG CCGCCTAAGG TAAAGAGAAA CATTGTTTTA TGAAGTG). SEQ ID NO: 32 is the nucleotide sequence ofthe PCR primer A657D Forward mutagenic internal (TCCAGCTGGG AATATTACGA CTCTGTCTAC ACAGAGAGAT T). SEQ ID NO: 33 is the nucleotide sequence ofthe PCR primer Reverse A657D mutagenic internal (AAT CTC TCT GTG TAG ACA GAG TCG TAA TAT TCC CAG CTG GA). SEQ ID NO: 34 is the nucleotide sequence ofthe PCR primer hFAP-RV-F (forward)

(TAG ATG GAA ATT ACT TAT GGT ACA AGA TGA TTC TTC C). SEQ ID NO: 35 is the nucleotide sequence ofthe PCR primer hFAP-Not-R (reverse) (GGT CGC TCA GCG GCC GCT TAGTC TGA CAA AGA GAA ACA CTG CTT TAG). SEQ ID NO: 36 is the nucleotide sequence ofthe PCR primer Y124H-F (TTTGTATATC TAGAAAGTGA TTATTCAAAG CTTTGGAGAC ACTCTTACACA G). SEQ ID NO: 37 is the nucleotide sequence of the PCR primer A207S-R (CCA GAG

AGC ATA TTT TGT AGA AAG CAT TTC CTC TTC). SEQ ID NO: 38 is the nucleotide sequence ofthe PCR primer A207S-F (GAAGAGGAAATGCTTTCTACAAAATATGCTCTCTGG). SEQ ID NO: 39 is the nucleotide sequence ofthe PCR primer hFAP-Cla-F (CGG ATA TTT ATT ATC GAT ACC ACT TAG CCT GC). SEQ ID NO: 40 is the nucleotide sequence ofthe PCR primer R356-R. primer (TGA AGG CCT AAA TCT TCC AAC CCA TCC AGT TCT GCT TTC TTC TAT ATGCTCC) SEQ ID NO: 41 is the nucleotide sequence ofthe PCR primer R356-F (TGGGTTGGAA GATTTAGGCC TTCAACACC AGTTTTCAG CTATGATG) SEQ ID NO: 42 is the nucleotide sequence ofthe PCR primer hFAP-RV-R

(CTGTATTTGCTGTTAAT TGG ATA TCTTACCTTGCAAGCACAGAAAACATT). SEQ ID NO: 43 is the nucleotide sequence ofthe PCR primer hFAP-RV-F (TAGATGGAAA TTACTTATGG TACAAGATGA TTCTTCC). SEQ ID NO: 44 is the nucleotide sequence ofthe PCR primer DEDH-R (AATGTGGTAC TCTGACGAAG ACCACGGCTT ATCCGGCCTG T). SEQ ID NO: 45 is the nucleotide sequence ofthe PCR primer DEDH-F (TGGTCTTCGT CAGAGTACCA CATTGCCTGG). SEQ ID NO: 46 is the nucleotide sequence ofthe PCR primer pSecTag-R (GGCGCTATTC AGATCCTCTT CTGAGAT). SEQ ID NO: 47 is the nucleotide sequence ofthe PCR primer FAP-DPP4-RI-F

(GGATGATAAT CTTGAGCAC TATAAGAATT CAACAGTCAT GAGCAGAGCT). SEQ ID NO: 48 is the nucleotide sequence ofthe PCR primer DPP-FAP-R (AGG CCG GAT AAG CCA TGG TCT TCA TCA GTA TAC CAC ATT GCC TGG A). SEQ ID NO: 49 is the nucleotide sequence ofthe PCR primer DPP-FAP-F (CAATGTGGTA TACTGATGAA GACCATGGCT TATCCGGCCT GTCCAC). SEQ ID NO: 50 is the nucleotide sequence ofthe PCR primer DPP4-A663-F (TCC CGG TGG GAG TAC TAT GCC TCA GTG TAC ACA GA). SEQ ID NO: 51 is the nucleotide sequence ofthe PCR primer DPP4-A663-R (TCT GTG TAC ACT GAG GCA TAG TAC TCC CAC CGG GA). SEQ ID NO: 52 is the nucleotide sequence ofthe PCR primer DPPIV 1300-F (AAGACTGCAC ATTTATTACA AAAGGCACC). SEQ ID NO: 53 is the nucleotide sequence ofthe PCR primer Swal-F

(GACATTTATG ATTTAAATAA AAGGCAGCTG ATTAC AGAA GAG). SEQ ID NO: 54 is the nucleotide sequence ofthe PCR primer R356-R (CTG AAG CGA AAA AAC CTC CAG CCC AGC CAG TAG TAC TCA TTC AAT G). SEQ ID NO: 55 is the nucleotide sequence ofthe PCR primer R356-F (GCTGGAGGTT TTTTCGCTTC AGAACCTCAT TTTACCCTTG ATGGT). SEQ ID NO: 56 is the nucleotide sequence ofthe PCR primer DPPIV_BspEI-R sequencing primer (TAG TAC TGA CAC CTT TCC GGA TTC AGC TCA). SEQ ID NO: 57 is the nucleotide sequence ofthe PCR primer H124Y-R (GCCTTTTATTTAAAT CAT AAA TGT CAT ATG AAG CTG TGT AGG AAT aCC TCC ATT). SEQ ID NO: 58 is the nucleotide sequence ofthe PCR primer S209A-R (ACC ACC ACA GAG CAG CGT AGG CAC TGA AGA CT). SEQ ID NO: 59 is the nucleotide sequence ofthe PCR primer S209A-F (AGTCTTCAGT GCCTACTATG CTGTGTGGTG GT). SEQ ID NO: 60 is the nucleotide sequence ofthe PCR primer mFAP45 (TTC CAT

TGG GCC CAC GTG GTG). SEQ ID NO: 61 is the amino acid sequence of a human soluble FAP alpha dimer enzyme (corresponding to SEQ ID NO:2 minus N-terminal amino acids 1-26). SEQ ID NO: 62 is the consensus amino acid sequence from the alignment of hFAP dimerization region 1 and hDPP4 dimerization region 1. SEQ ID NO: 63 is the consensus amino acid sequence from the alignment of hFAP dimerization region 2 and hDPP4 dimerization region 2. SEQ ID NO: 64 is the DNA sequence corresponding to the hFAP sequence shown in Figure 1. SEQ ID NO: 65 is the nucleotide sequence ofthe Sfi-FAP-B primer that encodes SEQ

ID NO:25. SEQ ID NO: 66 is the amino acid sequence corresponding to the hDPPIV sequence shown in Figure 2. SEQ ID NO: 67 is the nucleotide sequence corresponding to the hDPPIV sequence of GenBank accession number NM_001935. SEQ ID NO: 68 is the nucleotide sequence corresponding to the murine DPPIV sequence of GenBank accession number NM_010074. SEQ ID NO: 69 is the amino acid sequence corresponding to the murine DPPIV sequence of GenBank accession number NM_010074. SEQ ID NO: 70 is the amino acid sequence of a human soluble FAP alpha dimer enzyme (corresponding to SEQ ID NO:2 minus N-terminal amino acids 1-37).

Brief Description of the Drawings FIG. 1 illustrates the amino acid and corresponding coding nucleotide sequences for each monomer that contributes to wild type human FAP alpha dimer enzyme. FIG. 2 is an alignment ofthe amino acid sequences of each monomer that contributes to wild type human FAP alpha dimer enzyme and human DPPIV. FIG. 3 A is a bar graph showing the requirement of IL-1 signaling for chemokine and cytokine responses to PT-100. FIG. 3B is a bar graph showing the requirement of IL-1 signaling for chemokine and cytokine responses to PT- 100. FIG. 4 A is a graph showing G-CSF response to PT-100 in mice is undiminished in the absence of CD26 in vivo. FIG. 4B is a graph showing TARC response to PT-100 in mice is undiminished in the absence of CD26 in vivo. FIG. 4C is a graph showing KC response to PT-100 in mice is undiminished in the absence of CD26 in vivo. FIG. 4D is a graph showing MIP-1 beta response to PT-100 in mice is undiminished in the absence of CD26 in vivo. FIG. 4E is a graph showing eotaxin response to PT-100 in mice is undiminished in the absence of CD26 in vivo. FIG. 5 is a bar graph showing the level of soluble FAP alpha dimer enzyme produced from transfected 293T cells. FIG. 6 is a bar graph showing the ability of F19 antibody to recognize and bind to soluble human, but not mouse, FAP alpha dimer enzyme from culture supernatants of transfected 293T cells. FIG. 7 is a graph showing inhibition of soluble human FAP alpha dimer enzyme by PT-100. FIG. 8 is a graph comparing enzyme inhibition of soluble human FAP alpha dimer enzyme and native human FAP alpha using PT-100. FIG. 9 shows of soluble secreted FAP alpha dimer enzyme activity in tissue culture supernatant from plasmids #23, #29 and #43 measured by production of fluorescence from Ala-Pro- AFC substrate at pH 8.1. FIG. 10 shows FAP alpha dimer enzyme and DPPIV activity in several harvests of tissue culture supernatant from plasmids #122 and #135 respectively. FIG. 11 shows results of inhibition of FAP alpha dimer enzyme from plasmid #217, 219, 251, 255, 257, 233 and 245 by Val-nitriloPro compared to FAP alpha dimer enzyme (#122) and DPPIV (#135). FIG. 12A shows the pH activity profile of FAP alpha dimer enzyme comprising the A657D amino acid substitution (plasmid #233). FIG. 12B shows the IC50 of FAP alpha dimer enzyme comprising the A657D amino acid substitution (plasmid #233) for val-boroPro. FIG. 12C shows the binding kinetics of Val-boroPro to the FAP alpha dimer enzyme comprising the A657D amino acid substitution (plasmid #233). FIG. 12D shows the binding kinetics of Val-boroPro to the FAP alpha dimer enzyme encoding in plasmid #122. FIG. 12E shows the activity versus Ala-Pro-AFC substrate concentration for Km determination of human FAP alpha dimer enzyme from plasmid 122. FIG. 12F shows the activity versus Ala-Pro-AFC substrate concentration for Km determination of human FAP alpha dimer enzyme comprising the A657D amino acid substitution (plasmid 233). FIG. 13A shows the pH activity profile of DPPIV mutant comprising the D663A amino acid substitution (plasmid #266). FIG. 13B shows the IC50 of DPPIV mutant comprising the D663A amino acid substitution (plasmid #266) for Val-boroPro with simultaneous addition of substrate and inhibitor. FIG. 13C shows the binding kinetics of Val-boroPro to the DPPIV mutant comprising the D663A amino acid substitution (plasmid #266). FIG. 13D shows the binding kinetics of Val-boroPro to wild type human DPPIV (plasmid #135). FIG. 13E shows the activity versus Ala-Pro-AFC substrate concentration for Km determination ofthe DPPIV mutant comprising the D663A amino acid substitution (plasmid #266). FIG. 14 shows the IC50 determination of hFAP alpha dimer enzyme with Q732E and N733D amino acid substitutions (plasmid #94) compared to wild-type FAP alpha dimer enzyme (plasmid #122) for Val-boroPro and Val-nitriloPro inhibitors. FIG. 15 shows Eadie-Hoftsee plots for determination of Km for FAP-DPPIV chimera produced from plasmid #155 in tissue culture supernatant compared to control FAP alpha dimer enzyme (#122). The gradient is the negative value ofthe Km. FIG. 16A is a series of maps of plasmids encoding representative human-mouse chimeras of soluble FAP alpha dimer enzyme. FIG. 16B is a bar graph showing the relative activity of tissue culture supernatants of representative human-mouse chimeras of soluble FAP alpha dimer enzyme. FIG. 16C is a bar graph showing the relative activity of tissue culture supernatants of representative human-mouse chimeras of soluble FAP alpha dimer enzyme.

It is to be understood that the Figures are not required for enablement ofthe invention.

Detailed Description of the Invention The invention provides compositions such as pharmaceutical preparations comprising FAP alpha dimer enzyme, as well as methods of using such compositions in order to modulate immxxne responses. The invention is premised, in part, on the observation that treatment of bone marrow derived stromal cells in vitro with the boronic dipeptide, Val-boroPro⁴⁰ (PT-100), has been shown to increase the levels of IL-1 beta in tissue culture supernatants after several hours of incubation (see Example 1). Both FAP and CD26 can be detected in bone marrow derived stromal cell isolated in tissue culture. However, the effect of PT-100 appears to result from the inhibition of FAP and not CD26 because IL-1 beta levels increased in response to PT-100 in cultures of bone marrow stromal cell derived from Fischer D^~ rats bearing a mutation ofthe CD26 gene⁴¹ (see Example 1) and also in spleens of mice with a knockout in the CD26 gene (see Example 1.2). Accordingly, FAP alpha dimer enzyme appears to restrain the production of IL-1 because the production of IL-1 has been found to be increased in vitro and in vivo when the enzymatic activity of FAP alpha dimer enzyme is inhibited, as described herein. FAP alpha dimer enzyme possesses dipeptidyl peptidase activity, and inhibition of this activity with PT- 100, either in a culture system containing bone marrow derived stromal cells or in vivo in mice, caused significantly increased IL-1 production in numerous experiments. If IL-1 production can be increased by blockade of FAP alpha dimer enzyme, it follows that IL-1 production should be reduced by an increase in the level of FAP alpha dimer enzyme. As will be discussed in greater detail below, FAP alpha dimer enzyme encompasses wild-type and mutant FAP alpha dimer enzymes, membrane bound as well as soluble FAP alpha dimer enzymes, heterodimers comprising FAP and a second, related molecule such as DPPIV, and the like. Thus, as will be discussed below, the invention contemplates in a general sense methods for down-modulating an immune response. In most instances, the immune response is an abnormal immune response, an example of which is hyperimmunity. In some instances, immune response down-modulation can result in the treatment or amelioration of a particular condition associated with the immune response. Down-modulation is effected by increasing the level of FAP alpha dimer enzyme (and as a result FAP alpha dimer enzymatic activity) in a subject.

FAP alpha dimer enzyme: The invention contemplates the use of FAP alpha dimer enzyme. As used herein, "FAP alpha dimer enzyme" refers to a protein having FAP alpha dimer activity. Wild type FAP alpha dimer has been reported to possess a number of activities including dipeptidyl peptidase activity, collagenase/gelatinase activity, and extracellular matrix degradation activity. Any of these activities may be used to screen putative FAP alpha dimer enzymes for use in the methods ofthe invention. An example of a dipeptidyl peptidase assay for tracking FAP alpha dimer enzymatic activity is provided in the Examples. Collagenase/gelatinase activity and extracellular matrix degradation activity can be assayed as described by Aoyama A et al. 1990. Proc Natl Acad Sci USA. 87:8296-300; Monsky WL et al. 1994. Cancer Res. 54:5702-10. As used herein "FAP alpha dimer enzyme activity" refers to at least the dipeptidyl peptidase activity of wild type FAP alpha dimer enzyme. All mutations described herein (particularly with respect to FAP alpha dimer enzyme amino acid sequence) are relative to human wild type amino acid sequence provided as SEQ ID NO: 2. In addition, the aligned human and mouse wild type FAP amino acid sequences have the same numbering (as used herein) up to residue 736 out of a total of 760 (human) or 761 (mouse) amino acids. Recombinant FAP has reportedly been produced by two methods in the prior art. Firstly, full-length cell membrane bound recombinant FAP has been expressed in mammalian cell lines³⁵ and in insect cells with an additional N-terminal His-tag (Sun et al. 2002, Protein Expr. Purif. 24, 274-281). Full-length membrane-bound enzyme has several disadvantages in that detergents are needed for its extraction from the cell membrane. Detergents present in the solubilized material are undesirable at least because they are not typically pharmaceutically acceptable. In addition, removal of detergent generally triggers undesirable agglutination or the formation of higher order oligomers due to the presence ofthe hydrophobic membrane-spanning domain. In contrast, soluble FAP alpha dimer enzyme avoids these limitations. Secondly, a soluble recombinant chimeric CD8-mouse FAP protein form has been reported consisting of mouse CD8 residues 1-189 and FAP residues 27-761 (ref. 35). The CD8 portion forms a disulfide bond, which serves to keep the FAP dimerized. The first 26 amino acids of FAP including the transmembrane domain were removed. Whereas this form is soluble and avoids the use of detergents, it has the disadvantage that each monomer molecule is significantly larger by an extra 162 amino acids relative to wild type FAP alpha dimer enzyme, and that properties ofthe added sequence, for example binding to CD8 ligands, may perturb activity or localization ofthe soluble enzyme. Also, expression ofthe fusion in an insect cell line may have altered the glycosylation residues in an uncontrolled fashion, which is significant since the activity of FAP alpha dimer enzyme towards some substrates, notably gelatin, appear to be influenced by glycosylation. The compositions provided by the invention demonstrate that the CD8-mediated dimerization is dispensable since stable soluble FAP alpha dimer enzyme can be made without addition of extraneous means of dimerization as described in the Examples.

Wild type: As used herein, wild type FAP alpha dimer enzyme refers to full length FAP alpha dimer enzyme which is comprised of two wild type monomeric units. Full-length cDNA for FAP alpha monomer has been cloned previously. The human wild type FAP alpha monomer has an amino acid sequence of SEQ ID NO: 2 as derived from GenBank Accession Number NM_004460. The amino acid sequence (and corresponding coding nucleotide sequence) of the monomeric units that contribute to wild type liuman FAP alpha dimer enzyme are shown in FIG. 1. Wild type FAP alpha dimer enzyme is an integral cell surface membrane protein having a cytoplasmic domain (amino acids 1 to 6 in SEQ ID NO: 2), a transmembrane domain (amino acids 7 to 26 in SEQ ID NO: 2), and an extracellular domain (amino acids 27 to 760 in SEQ ID NO: 2). These various regions as well as other structural features of wild type FAP alpha dimer enzyme are illustrated in FIG. 2, which provides an alignment of amino acid sequences of FAP alpha and DPPIV monomers. Wild type FAP alpha monomer is approximately 48% identical to wild type CD26 monomer at the amino acid level. FAP alpha and CD26 monomers form heterodimers. Full-length cell membrane bound recombinant FAP has been expressed in insect (Sun et al. 2002, Protein Expr. Purif. 24, 274-281) and mammalian cell lines³⁵. However, extraction from the membrane requires the addition of detergent to solubilize the membrane and the hydrophobic membrane-spanning region ofthe protein³⁵, which leads to undesirable contaminants. FAP does not appear to have a naturally occurring soluble counterpart, unlike CD26. The soluble form of CD26 results from apparent cleavage ofthe membrane-bound form. Rather FAP appears to exist as either a monomer or dimer (either with itself or with other proteins such as CD26). In its monomeric form it has a molecular weight of about 88 to about 95 kilodaltons according to SDS-PAGE, and is catalytically inactive in this form. In its dimeric form, it has a molecular weight of about 170 kilodaltons according to SDS-PAGE. FAP alpha dimer enzyme may comprise the catalytic domain of wild type FAP alpha dimer enzyme, in whole or in part. The catalytic αβ hydrolase domain of wild type FAP alpha dimer enzyme is present at amino acids 500-760 of SEQ ID NO: 2. Activity however appears dependent upon the presence ofthe β propeller domain as well, in whole or in part. The β propeller domain is present at amino acids 55-499 of SEQ ID NO: 2.

Improved forms of FAP: FAP alpha dimer enzyme encompasses various other protein forms that possess FAP alpha dimer activity. These include but are not limited to truncated versions ofthe wild type protein, chimeric proteins comprising regions of other proteins grafted internally into the FAP alpha sequence, fusion proteins comprising sequence from wild type FAP alpha dimer enzyme conjugated (directly or indirectly) to sequence from one or more other proteins, fusion proteins comprising sequence from wild type FAP alpha dimer enzyme conjugated (directly or indirectly) to non-FAP alpha protein sequence (e.g., leader sequences, recombinant vector sequences, and the like), heterodimeric proteins comprising at least one monomer having FAP alpha enzymatic activity in association with another monomer, point mutants of wild type FAP alpha dimer enzyme, variants of FAP alpha dimer enzyme that comprise conservative amino acid substitutions relative to the wild type amino acid sequence, and the like. The commonality between FAP alpha dimer enzyme species is the retention of FAP alpha dimer enzymatic activity. FAP alpha dimer enzymes can have levels of enzymatic activity that are less than (but still therapeutically useful), equal to, or greater than wild type FAP alpha dimer enzyme. In some embodiments, the enzymatic profile of FAP alpha dimer enzyme is different from wild type either in the substrate affinity, the pH sensitivity, and the like. Examples of FAP alpha dimer enzymes include N-terminal truncations and deletions; internal point mutations, insertions and deletions; chimeras comprising FAP alpha monomers from different organisms and FAP-DPPIV chimeras; heterodimeric and monomeric forms; and inactive forms that regain activity over time. Point mutations, deletions, insertions or chimeras that alter local charge, protein solubility, stability, biological half-life, interactions with other proteins, formulation properties, shelf-life or other such property are also contemplated by the invention. Mutations to wild type FAP alpha dimer enzyme can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Additional methods are described in the Examples. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene encoding a FAP alpha dimer enzyme. The substitutions also can be made by directly synthesizing the protein or a fragment thereof. The activity of putative FAP alpha dimer enzymes can be tested by cloning the nucleic acid encoding the putative enzyme into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the putative enzyme, and testing for FAP alpha dimer enzymatic activity, as described herein. Such screening strategies are described in greater detail in the Examples. Under certain circumstances of use, it is beneficial to introduce or enhance various desirable properties into a FAP alpha dimer enzyme. These altered properties include but are not limited to singly or in combination: altered Km for substrate (i.e., lower or higher Km); differential changes in Km values for specific substrates leading to altered substrate specificity and /or selectivity; a more rapid rate of substrate turnover or rate of overall catalysis; a less restrictive pH profile or broader pH optimum; an altered (e.g., lower or higher) IC50 inhibition constant for certain inhibitors; the ability to be inactivated or essentially irreversibly inhibited by a known inhibitor; altered dimerization properties; altered gelatinase activity; altered thermal stability; altered biological half-life in serum; and the like. Another possible beneficial property is the ability to be inhibited by inhibitors containing the nitrilo group including nitrilo-analogues of proline boronic acid inhibitors in which the boronic acid moiety is replaced by a nitrilo moiety (2-nitrilo-pyrollidines), including nitrilo- analogues of X-boroPro inhibitors where X = a natural or unnatural L- or D- amino acid such as valine boroPro. These nitrilo inhibitors are capable of inhibiting DPPIV (Ashworth, D.M. et al. Biorganic and Medicinal Chemistry Letters. 2-cyanopyrrolidines as potent, stable inhibitors of dipeptidyl peptidase IV. 1996. vol. 6, 1163-1166), but by comparison are less effective against wild-type FAP (vide infra). Truncated and soluble forms: FAP alpha dimer enzymes are preferably soluble in nature. One approach to generating a soluble FAP alpha dimer enzyme is to truncate the extracellular domain. Accordingly, some species of FAP alpha dimer enzyme corresponds to truncated forms ofthe wild type protein. Preferably, these forms are truncated at the N-terminus and include truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. For example, SEQ ID NO: 61 lacks amino acids 1-26 of hFAP. Deletion ofthe first 37 amino acids in SEQ ID NO: 2, in whole (e.g., SEQ ID NO: 70 , which lacks amino acids 1-37) or in part, can be made without significant loss to activity. The Examples elaborate on such FAP alpha dimer enzyme forms. These truncated forms may include the cytoplasmic domain and the extracellular domain fused to each other, and thus would lack the transmembrane domain. Alternatively, they may lack both the cytoplasmic and transmembrane domain. These latter forms, as well as other forms described herein, may further comprise additional amino acids that are not derived from wild type FAP alpha dimer enzyme. Truncations at the N terminus are preferred. Soluble forms of FAP alpha dimer enzyme are also contemplated by the invention. As used herein, a soluble FAP alpha dimer enzyme is a FAP alpha dimer enzyme that is not cell membrane associated. Soluble FAP alpha dimer enzyme can be made by removing part or all ofthe transmembrane domain (as described above), and optionally fusing a secretory signal sequence to it to effect secretion ofthe protein outside the cell. The Examples describe the generation of a soluble FAP alpha dimer enzyme which lacks the wild type FAP alpha dimer enzyme N-terminal sequence which is involved in anchoring the protein to the cell membrane. In one such embodiment, the N-terminus ofthe resulting FAP alpha dimer enzyme therefore starts at any residue between amino acid 25-38 inclusive of wild type human FAP alpha dimer enzyme based on numbering in SEQ ID NO: 2 that starts at the proposed methionine start codon. The soluble FAP alpha dimer enzyme retains enzymatic activity, as shown in the Examples. In another embodiment, a soluble version of FAP alpha dimer enzyme is made in which the transmembrane domain is wholly or partially deleted, or some of its residues mutated to more hydrophilic ones thereby preserving its native N-terminus, but abrogating the membrane localization ofthe protein and rendering it soluble. Fusions: A FAP alpha dimer enzyme can be a fusion protein of FAP alpha dimer enzyme sequence conjugated to non-FAP alpha amino acid sequence. "Non-FAP alpha amino acid" refers to amino acid sequence that does not exist in wild type FAP alpha dimer enzyme. It may however exist in other known proteins (i.e., it may not be a random sequence). Non-FAP alpha amino acid sequences may comprise one or more amino acid residues. Examples of non-FAP alpha amino acid sequences include amino acid sequences of a non-FAP alpha protein domain (in whole or in part), a signal or leader secretion sequence (e.g., a G-CSF leader sequence), a purification domain or sequence (e.g., a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, or a maltose binding domain), an epitope (e.g., a hemaglutimiin tag, a FLAG tag, a V5 tag, a myc tag, or a T7 tag), a linker, a protein degradation sequence (e.g., a PEST sequence), a protease cleavage site, a self-cleaving affinity tag (e.g. Intein 1 & intein 2), a tissue localization sequence, a peptide or protein ligand that targets the protein to particular cell surface molecules, and the like. In yet other embodiments, the non-FAP alpha tag may provide a suitable cleavage site to produce a specified N-terminal amino acid after cleavage or to facilitate removal ofthe non-FAP portions (e.g. a purification domain or sequence or an epitope tag) after for example purification. Examples of proteases suitable for cleavage of recombinant FAP dimer include enterokinase, factor Xa protease, thrombin, TEV protease, PreScission protease, Furin,

Genenase as described by LaVallie et al (1994). In Current Protocols in Molecular Biology. pp. 16.4.5-16.4.17, John Wiley and Sons, Inc, New York, NY; Stevens Structure, 8: R177- R185 (2000); Cameron, A. et al. (2000) J. Biol. Chem. 275, 36741-36749; Krysan DJ et al. J Biol Chem. 1999 274, 23229-34; and Carter, P. et al. (1989) Proteins: Structure, Function, and Genetics 6, 240-248. Examples of target molecules for peptide ligands include integrins, intercellular adhesins, addressins, various GPI-linked molecules, C-type and other lectins, cytokine and chemokine receptors and the like. Protein or epitope sequence may also be attached at either the N- or C-terminus to aid purification, localization or detection ofthe FAP alpha dimer enzyme. As described above, the various tags may also be attached via a sequence that provides a proteolytic site for removal of said tag after purification. In other embodiments, a tag is attached at the N- or C-terminus to enhance therapeutic efficacy ofthe FAP alpha dimer enzyme. Non-FAP alpha amino acid sequences may be fused upstream ofthe first FAP- derived residue or to the C-terminal ofthe protein. The resultant protein may be expressed either cytoplasmically or secreted, with secretion made possible by fusion to a suitable secretion sequence. The fusion point between FAP and secretion sequences preferably allows for cleavage ofthe secretion sequences to give the desired FAP-derived N-terminal amino acid in the mature secreted form. Secretion sequences typically are derived from type I transmembrane proteins or secreted proteins such as immunoglobulins, serum proteins, hormones, chemokines, cytokines, certain cytokine receptors which have single membrane spanning domains and the like. An example of a commercially available secretion vector for mammalian expression is pSecTag2B vector (InVitrogen Corporation). The use of some vectors may add one or more N-terminal amino acids to the mature cleaved protein depending on the relative location ofthe cloning site and the site of leader cleavage. Thus in one embodiment, a signal sequence derived from a cytokine (e.g., G-CSF) or chemokine is fused to the FAP monomer nucleotide sequence. Examples of cytokine and chemokine genes providing leader sequences for production of secreted soluble FAP alpha dimer enzyme and DPPIV include the following: Interleukins 2, 3,4,5,6,7,8,9,10,11,12,13,15, 16, 17 and the like; cytokines such as G-CSF, GM-CSF, TGF, Tpo; chemokines from both the C-C and CXC families including MCP, MIP- lalpha, MlP-lbeta, ENA-78, eotaxin, HCC-1, RANTES, TARC and also the CXXXC family exemplified by Fractaline (neurotatin). Sequences for these various factors can be found in publications such as "The Cytokines: Facts Book" Fitzgerald et al. Academic Press, ISBN 0- 12-155142-3. Examples of cytokine receptor secretion sequences include IL-1 Type I and Type II receptors, IL-2 receptor alpha, beta or gamma chain, IL-3 alpha and IL-3 beta receptor subunits and the like, the sequences of which can be found in publications such as "The Cytokines: Facts Book" Fitzgerald et al. Academic Press, ISBN 0-12-155142-3. It is understood that cytoplasmic versions of FAP alpha dimer enzymes are also embraced by the invention. In these embodiments, the cells are lysed and the soluble form of FAP alpha dimer enzyme is released and can be further isolated.

Chimeras: As used herein with respect to FAP alpha dimer enzymes, the term "chimera" means a dimer comprised of two non-identical monomers, or a dimer comprised of at least one monomers that itself is derived from at least two different sources. The monomers could derive from different proteins, or from the same protein but different species. Examples of chimeras contemplated by the invention include FAP human-mouse chimeras made by splicing human and mouse FAP segments, and FAP-DPPIV chimeras. The latter category of chimeras can be further subdivided into (1) chimeras that are substantially DPPIV-like structurally and immunologically but with FAP-like catalytic or other enzymatic properties, (2) chimeras that are substantially FAP-like (especially enzymatically) but with some amino acid residue substitutions from DPPIN, and (3) chimeras that resemble both parent molecules in approximately equal proportions. In the case of FAP -DPPIV chimeras, the chimera may contain at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% FAP residues. In important embodiments, the chimera comprises the catalytic domain of wild type FAP alpha dimer enzyme either in whole or in part. It alternatively comprises substitutions, additions or deletions in the catalytic domain that do not abrogate FAP alpha dimer enzymatic activity. Another preferred embodiment is FAP alpha dimer enzyme or a protein that is substantially wild type FAP alpha dimer enzyme structurally and immunologically, but with some DPPIV-like catalytic properties including but not limited to inhibitor specificity, pH profile and other properties beneficial to the therapeutic use, or to the modulation of its therapeutic properties. Yet another preferred embodiment is DPPIV or a protein that is substantially DPPIV structurally and immunologically, but with FAP-like catalytic or other enzymatic properties.

Mutations to the catalytic domain: FAP alpha dimer enzymes also embrace proteins having mutations in the residues lining the interior ofthe active site cavity. Amino acid residue changes which confer altered properties include changes in (1) active site residues involved in substrate binding or catalytic events or the internal surface ofthe active site cavity (e.g., amino acids L48-S63, L89-V98, Ν102-P107, S116-Y126, Q151-P157, Q167-L172, W199-P216, M285-T300, G345-S357, I367-G373, W395-Q405, Y410-N413, Y450-D457, Y462-Y467, 1538-1558, A578-D582, L592, W621-V629, V647-V650, Y656-D657, V659-T661, Y677, D703-V705 and H733- L735) , (2) residues lining the entrance to the active site which are known to exert electrostatic or steric influence on substrate binding or product release in enzymes described below, or (3) residues that confer structure to the active site activity or entrance such that FAP alpha dimer enzyme conformed more closely to substrates of interest or had altered biological or thermal stability. In addition, amino acid changes that affect the accessibility of water molecules would be predicted to affect solvation of substrates or products and influence reaction kinetics. One approach to mutating wild type FAP alpha dimer enzyme is to compare its structure and sequence with a homologous enzyme such as DPPIV in an attempt to import DPPIV properties without substantially altering key desired characteristics of FAP alpha dimer enzyme. DPPIV is the closest known homolog of wild type FAP alpha dimer enzyme and it has some properties that would be desirable in FAP alpha dimer enzyme. There are over 372 amino acid differences between the two enzymes, with an even larger number of possible combinations of residues if multiple interacting amino acid residues define a particular property. Desirable DPPIV properties include thermal stability, more rapid kinetics, long serum half-life, the ability to be efficiently inhibited by inhibitors containing the nitrilo group, and the like. In one approach, residues lining the active site cavity of DPPIV are identified by scanning the interior ofthe DPPIV crystal structure (atomic coordinate file PDB #lnlm from Protein Data Bank) adjacent to the bound inhibitor, using the Protein Explorer freeware. The active site cavity in DPPIV is large and the same is assumed for FAP alpha dimer enzyme. Active site cavity residues include regions ofthe beta propeller lining the internal channel from the apical opening, and certain residues from the C-terminal 200 amino acid portion suggested to constitute the putative catalytic domain prior to the crystal structure determination. Together, they include DPPIV amino acids L49-S64, L90-F98, 1102-P109, Y118-Y128, Q153-P159, N169-V174, W201-P218, S292-T307, G352-T365, 1374-G380, W402-S412, Y417-N420, Y456-K463, Y468-S473, L544-A564, G584-D588, L598, W627- V635, V653-V656, Y662-D663, V665-T667, Y683, D709-V711 and H740-A743. By analogy, the corresponding residues of wild type FAP alpha dimer enzyme can be identified from an alignment ofthe two sequences. Therefore, in FAP alpha dimer enzyme, residues lining the internal cavity include L48-S63, L89-V98, N102-P107, SI 16-Y126, Q151- P157, Q167-L172, W199-P216, M285-T300, G345-S357, 1367-G373, W395-Q405, Y410- N413, Y450-D457, Y462-Y467, 1538-1558, A578-D582, L592, W621-V629, V647-V650, Y656-D657, V659-T661, Y677, D703-V7Q5 and H733-L735. It is reasonable to assume that these residues are located in a similar relative position in wild type human FAP alpha dimer enzyme. Differences in putative binding-site residues are candidates for targeted changes aimed at importing attributes from DPPIV into FAP alpha dimer enzyme and vice versa. Mutation of these residues in FAP alpha dimer enzyme to alter substrate recognition, hydrolysis properties, inhibition profiles or stability are important embodiments. Some of these residues are noted in FIG. 2 which shows an alignment of monomer amino acid sequences from wild type human FAP alpha dimer enzyme and wild type human DPPIV. These residues include H126, S209, V354-P359 inclusive, and D663 in DPPIV and Y124, A207, A347, G349, F351, V352 and A657 in wild type hFAP alpha dimer enzyme. The resultant changes therefore correspond to Y124H, A207S, A347V, G349R, F351R, V352P and A657D in hFAP alpha dimer enzyme and H126Y, S209A, V354A, R356G,R358F, P359V and D663A in DPPIV. FAP alpha dimer enzymes may comprise one of more of these mutations. FAP alpha dimer enzymes comprising mutation of any ofthe other amino acid residues contributing to the internal surface (e.g., R421, S548, R550, in particular R421G, S448K and R550D substitutions), and ofthe corresponding residues in DPPIV are also embodiments in the current invention. Preferably, the catalytic triad residues consisting of S624, D702 and H734 in wild type FAP alpha dimer enzyme and S630, D708 and H740 in DPPIV are not mutated. Similarly, alterations in conserved (i.e., identical) residues that retain enzymatic activity are also preferred embodiments. By convention, amino acid substitutions are written in an abbreviated form such as for example Y124H, denoting substitution of tyrosine number 124 with histidine. Prior research has been published on aspartate residues conserved in the presumptive catalytic domain between human and mouse DPPIV. Mutations in mouse DPPIV established that aspartate 702 is part ofthe catalytic triad, and that mutation ofthe conserved Asp 599 and 657 residues (corresponding to D605 and D663 in human) to either alanine or threonine (D599) or glycine (D657) reportedly did not affect the enzymatic properties ofthe mouse enzyme. As a result, it was concluded that Asp657 mutations did not significantly modify the expression or enzymatic properties ofthe corresponding proteins. (David, F. et al. 1993. J. Biol. Chem., 268, 17247-17252.). Therefore, based on the prior art, human DPPIV residue D663 (mouse D657) is an unlikely candidate for altering enzymatic properties. The homologous residue A657 is not however conserved in wild type human FAP dimer enzyme. It has now been discovered according to the invention that mutation of residue A657 in FAP alpha dimer enzyme to its corresponding DPPIV amino acid (i.e., Ala to Asp) and mutation of residue D663 in DPPIV to its corresponding FAP alpha dimer enzyme amino acid (i.e., Asp to Ala) result in enzyme molecules that have new and unexpected properties, and account in large part for the observed enzymatic differences between the two enzymes. FAP alpha dimer enzyme can also comprise mutations in residues that line the opening ofthe active site. Mutation of these residues can alter the kinetics of substrate binding and catalysis. There are two putative openings to the active site, based on an analysis ofthe crystal structure of hDPPIV (Rasmussen et al. 2003, Nature Struc. Biol. 10, 19-25; Hiramatsu et al. 2003. Acta Crystallogr. D Biol Crystallogr. 59, 595-596, and Biochem. Biophys. Res. Comm. 2003. 302, 849-854; Haffmann et al. 2003. Proc. Natl. Acad Sci USA 100, 5063- 5068;Oefner et al. 2003. Acta Crystallogr. D Biol Crystallogr. 59, 1206-12; Thoma et al. 2003. Structure (Camb) 11, 947-959). One opening is located at the side ofthe molecule approximately near the interface ofthe beta-propeller domain and the catalytic domain. The other opening is located at the apex where the beta propeller loops come together. In human DPPIV, residues lining the side entrance to the active site are distributed in several regions of the primary sequence including amino acids 50-60, 72-75, 90-103, 116-127, and 740-746 in human, and 50-60, 70-73, 88-99, 111-121, and 734-740 in mouse. The corresponding human FAP alpha residues lining the side opening are approximately residues 49-59, 70-75, 90-103, 115-126, 735-741 (735-742 in mouse FAP). Examples of contemplated mutations therefore include N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q and Y124H. The apical opening is contributed by DPPIV residues S59-D65, S108-D110, S158- V160, S218-G220, T304-Q308, S360-D367, E408-D413, S458-A465 approximately and by inference the homologous FAP residues (dotted underline in FIG. 2). Examples of contemplated mutations include G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K and Y458E. Alterations in these and closely adjacent residues are preferred embodiments, including substituting DPPIV residues in FAP alpha dimer enzyme, as well as importing murine equivalents, e.g., GenBank Accession Number NM_010074 for wild type murine DPPIV (SEQ ID NOs: 68 and 69 for the nucleotide and amino acid sequences), GenBank Accession Number NM_001935 for wild type human DPPIV (SEQ ID NOs: 67 and 66 for the nucleotide and amino acid sequences), and GenBank Accession Number Y10007 for wild type murine FAP alpha dimer enzyme (SEQ ID NOs: 5 and 6 for the nucleotide and amino acid sequences), into the corresponding positions of human FAP alpha dimer enzyme and human DPPIV.

Glycosylation site mutations: FAP alpha dimer enzymes also include mutation at one or more ofthe six N-linked glycosylation sites in wild type human FAP alpha dimer enzyme. Four ofthe six potential N- linked glycosylation sites in human FAP (at amino acids 49, 92, 99, 227, 314, 679 ) are shared with hDPPIV (N49, N92, N314 and N679). DPPIV has 5 additional potential glycosylation sites. Introduction or deletion of individual glycosylation sites by site-directed mutation may affect critical properties including biological-half-life, thermal stability and gelatinase activity among others. However, it has been shown according to the invention that glycosylation at N227 is not required for FAP activity. That is, the invention embraces a FAP alpha dimer enzyme comprising a T229M mutation and that therefore lacks one ofthe six glycosylation sites by destroying the N-x-T glycosylation motif at N227. The invention however also embraces FAP alpha dimer enzymes that are wild type at residue 229. It has been reported that wild type FAP alpha dimer enzyme expressed in COS-1 cells is differently glycosylated than in human sarcoma and fibroblasts (Scanlan et al. 1994. Proc. Natl. Acad. Sci. 91, 5657- 5661), and it has further been reported that non-glycosylated FAP does not have gelatinase activity (Sun et al. 2002, Protein Expr. Purif. 24, 274-281). Thus, the FAP alpha dimer enzyme may further comprise mutations that result in the removal of one or more glycosylation sites to selectively reduce gelatinase/collagenase activity without impacting dipeptidyl peptidase activity. Compensatory additions of new glycosylation sites to preserve overall enzymatic or biological half-life, solubility and other desired properties, are also contemplated. Such compensatory additions include sites found in human DPPIV, surface lysines comprised of FAP alpha dimer enzyme residues 173, 191, 334, 372, 382, 436, 437, 445, 460, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, and 753; and K219T, or I192T which creates a N-x-T motif.

Disulfide bond mutations: DPPIV has 5 disulfide bonds, involving cysteine residues 328/339, 385/394, 444/447, 454/472 and 649/762 denoted in pairs. DPPIV is known to be stable at temperatures up to 55 °C. Wild type human FAP alpha dimer enzyme lacks equivalent Cys385 and Cys394 residues and thus has only 3 disulfide bonds (i.e., 6 ofthe 8 analogous cysteines of DPPIV are present). Disulfide bonds have been reported to contribute to stability in many secreted enzymes, examples of which are trypsin, chymotrypsin, lysosyme, ribonuclease and others. FAP alpha dimer enzymes may include addition of novel cysteines to potentiate disulfide bond formation in order to enhance protein stability or removal of disulfides to decrease stability depending on the therapeutic application. Examples include introduction ofthe cysteines equivalent to DPPIV Cys 385 and Cys394 into FAP alpha dimer enzyme by simultaneously mutating residues at or near H378 and A386 to cysteine (e.g., H378C and A386C). In another embodiment, a disulfide bond is introduced to secure the N-terminus to one ofthe C-terminal residues . This is accomplished by replacement of a pair of residues that are roughly juxtaposed in the tertiary structure with cysteines. The latter then form disulfide bonds in the folded protein. One embodiment includes mutation to cysteine of one of T38 or M39 or a nearby residue and simultaneously one of N506, Q508 or H533 which tether the N- terminus of FAP alpha dimer enzyme to the C-terminal region (e.g., one of T38C, M39C, plus one of N506C, Q508C and H533C). In another embodiment, residue L48 or a nearby residue and residue N742 or a nearby residue in FAP alpha dimer enzyme are changed to cysteine for the purpose of introducing a disulfide link (e.g., L48C and N742C). Residues M683 and 1713 can also be mutated to be cysteines (e.g., M683C and I713C). In other embodiments, disulfide bonds within FAP alpha dimer enzyme are removed by eliminating one or both ofthe participatory cysteines to modulate the stability and thus activity and biological half- life ofthe protein. Preferably, pairs of cysteines are mutated thereby avoiding formation of inappropriate disulfide bonds involving the remaining cysteine residue as has been suggested in the art.

PEGylation: FAP alpha dimer enzymes may also be PEGylated (i.e., conjugated to polyethylene glycol) in order to increase biological half-life. Modification of proteins with polyethylene glycol (PEG) can be used to reduce the immunoreactivity, prolong the clearance time (biological half-life) and improve stability of proteins. (Inada Y et al. Trends Biotechnol. 1995 13:86-91.) PEG is generally attached to proteins at a epsilon amino group of surface lysine residues, and methods have been described for altering residues to lysine in a protein to increase the number of sites for attachment. (Hershfield MS et al. Proc Natl Acad Sci U S A. 1991. 88:7185-9.). Another method, exemplified in U.S. Patent No. 6,608,183, attaches PEG to cysteine groups that are naturally present or engineered into the protein sequence. In the embodiments contemplated here, surface residues of FAP alpha dimer enzyme including but not limited to lysine and cysteine residues are labeled with PEG. In another embodiment, surface residues which are not normally reactive to the PEG labeling reagents are altered by site directed mutagenesis to either lysine or cysteine to allow attachment of PEG. The location of potential surface residues can be determined by analogy to the DPPIV crystal structure or empirically. In one embodiment, one or more glycosylation sites in FAP alpha dimer enzyme or DPPIV (underline in FIG. 2) are replaced by PEG by altering any one ofthe outside residues ofthe asparagine-x-serine/threonine glycosylation motifs (x= any amino acid) to lysine or cysteine for PEG attachment. In some embodiments, selected surface lysines are changed to cysteines for cysteine-PEG links. A list of examples of surface lysine candidates includes lysines at positions 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, and 753 in wild type FAP alpha dimer enzyme. In others, surface arginines at positions 91, 148, 263, 323, 343 and 444 in wild type FAP alpha dimer enzyme are altered to permit PEGylation. In yet another embodiment, lysine groups are removed to prevent PEG attachment in locations not conducive to enzyme function or to reduce the number of potential attachment sites. A list of examples of surface lysine candidates includes lysines at positions 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, and 753 in wild type FAP alpha dimer enzyme. In the case of heterodimers or chimeras, examples of DPPIV surface lysine candidates include lysines 41, 50 , 56, 71, 139, 163, 175, 190, 250, 267, 391, 392, 399, 423, 433, 441, 463, 466, 489, 502, 512, 513, 523, 536, 538, 539, 554, 589, 615, 622, 648, 696, 721 and 760 which can be changed in any combination to arginine in one embodiment, and in another to a mixture selected from arginine, other charged amino acid (e.g., aspartate, gfutamate or histidine) or polar amino acid (e.g., glycine, serine, threonine, asparagine or glutamine), but not to asparagines in cases where it would create an asparagine-x-serine/threonine glycosylation motif. In another embodiment, DPPIV lysines which line the internal cavity are changed in any combination as follows: K71 is changed to glutamine, K463 to aspartate and K554 to serine to prevent PEG attachment. In another embodiment, DPPIV lysines 122, 258, 373 and 512; and FAP lysines 120, 254 and 366 which appear to be important structurally, are excluded from elimination. In yet another embodiment, DPPIV lysines 122, 258, 373 and 512; and FAP lysines 120, 254 and 366 are altered to arginine or histidine.

Heterodimers: FAP alpha dimer enzymes also encompass heterodimers comprising FAP monomers and other monomers. These heterodimers may be formed by co-expression of monomers in the same cell. Heterodimer formation can be facilitated by the presence of matched dimerization domains engineered into one or both monomers as described below.

Dimerization: FAP alpha dimer enzymes may also embrace mutation at residues involved in the obligate dimerization. In one such embodiment residues in two regions presumptively involved in dimerization, based on scanning the hDPPIV crystal structure are targeted. Region 1 corresponds approximately to hDPPIV residues P234-V254

(PLIEYSFYSDESLQYPKTVRV; SEQ ID NO: 7) which form a loop with additional extra- loop residues Y256-V262 also participating in the interface, and FAP alpha dimer enzyme residues P232-I250 (PVIAYSYYGDEQYPRTINI; SEQ ID NO: 8) which form a loop with additional extra-loop residues Y252-K258 also participating in the interface. Region 2 corresponds to DPPIV residues F713-D738 (FQQSAQISKA LVDVGVDFQA MWYTD; SEQ ID NO: 9) and FAP alpha dimer enzyme residues F706-D731 (FQNSAQIAKA LVNAQVDFQA MWYSD; SEQ ID NO: 10). Region 1 has eight and Region 2 has six amino acid differences between wild type human FAP alpha dimer enzyme and DPPIV. In addition, FAP alpha dimer enzyme has a 2 amino acid deletion in region 1 relative to DPPIV based on optimal alignment.

Region 1. hFAP: TDIPVIAYSYYGDE--QYPRTINIPYPKAGAKN 259 SEQ ID NO T++P+I YS+Y DE QYP+T+ +PYPKAGA N SEQ ID NO 62 hDPP4: TEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVN 263 SEQ ID NO 7

Region 2. hFAP: 69₂ VDYLLIHGTADDOTHFQNSAQIAKALVNAQVDFQAMWYSDQNHGL-SG STNHLYTHMTHFLKQCFSL 758 V+YLLIHGTADDNVHFQ SAQI+KAV+ VDFQAMWY+D++HG+ S + H+YTH +HF+KQCFS hDPPIV: 698 VEYLLIHGTADDNVHFQQSAQISKALVDVGVPFQAMWYTDEDHGIASSTAHQHIYTHMSHFIKQCFSL 765 hFAP is SEQ ID NO: 10; hDPPIV is SEQ ID NO: 9; and consensus is SEQ ID NO: 63 FAP alpha dimer enzymes can also be FAP-DPPIV chimeras in which the dimerization interfaces in the proteins, either wholly or in part, are exchanged between the proteins. For example, replacing human FAP alpha dimer enzyme dimerization region 1 with DPPIV region 1 introduces eight amino acid changes and adds a two amino acid insertion. Replacing wild type human FAP alpha dimer enzyme dimerization region 2 with wild type DPPIV region 2 introduces 6 amino acid changes. Examples include FAP-DPPIV chimeras in which, separately or together, P232-I250 of wild type FAP alpha dimer enzyme Region 1 or a portion thereof is replaced with P234-V254 of wild type DPPIV or some portion thereof; and F706-D731 of wild type FAP alpha dimer enzyme Region 2 or a portion thereof is replaced with F713-D738 of wild type DPPIV or some portion thereof. Another preferred embodiment includes analogous DPPIV-FAP chimeras in which, separately or together, some portion of P234-V254 of wild type DPPIV is replaced with P232-I250 of wild type human FAP alpha dimer enzyme and/or F713-D738 of wild type DPPIV is replaced with F706-D731 of wild type human FAP dimer enzyme, wholly or in part. Also envisioned is the introduction of cysteine residues in the dimer interface, which, depending on location, may allow inter- subunit di-sulfide bonds, or in another case, intramolecular di-sulfide bonds, either within or between dimerization Regions 1 and 2. Embodiments include mutation of residue T251 of wild type DPPIV to a cysteine (i.e., T251C) which is proximal to the T251 ofthe other chain ofthe dimer, and analogously mutation of residue T248 of wild type FAP alpha dimer enzyme to a cysteine (i.e., T248C) to induce an inter-subunit disulfide bond. It is to be understood that the resultant chimeric monomers can then be combined in a variety of ways provided the resultant dimer possesses FAP alpha dimer enzymatic activity, as described herein.

Monomeric forms of FAP: In another embodiment, the loops that constitute the dimerization domains (see FIG.

2) are deleted to yield monomers. Additionally, residues that form the dimer interface may be altered to reduce affinity for intermolecular interaction and thereby favor monomer over dimer forms. These latter alterations include changing hydrophobic residues to hydrophilic residues to facilitate aqueous exposure. These changes may accompany other alterations to render the protein monomeric. Other embodiments target residues 252-262 of DPPIV and their FAP equivalents and mutation of DPPIV Y661 and FAP Y655 to hydrophilic residues such as aspartic acid, glutamic acid, asparagine, glutamine, serine, lysine, arginine, and histidine to allow solvent exposure.

Mutation of charged amino acid surface residues: FAP alpha dimer enzymes may also comprise mutation of charged amino acid surface residues. Such mutations may change the electrostatic properties ofthe enzyme. For example, crystallographic studies reportedly show that the residues of wild type DPPIV facing the membrane are positively charged, presumably to complement the negative charge of membrane lipids. A similar phenomenon may apply for FAP alpha dimer enzymes.

However, no such constraint need be applied to a FAP alpha dimer enzyme that is soluble (i.e., not membrane bound) and thus changes to exposed residues would be more tolerated. These exposed residues include surface lysines at positions 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, and 753; and surface arginines at positions 91, 142, 148, 175, 263, 323, 343, 444, 530 and 691. Mutation of arginines 109, 303 and 426 is also possible.

Conservative substitutions: The skilled artisan will also realize that FAP alpha dimer enzymes also comprise conservative amino acid substitutions relative to wild type sequence. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution which does not substantially alter the relative charge in one instance, or in another retains a charge but of opposite sign, or size characteristics ofthe polypeptide in which the amino acid substitution is made. Exemplary conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D (h) M, I, L, V, F, Y; and (i) K, R, H S, Q, N, E, D.

Inhibited forms of FAP and DPPIV which slowly recover activity: The invention further contemplates FAP alpha dimer enzymes that are reversibly inhibited by inhibitors such as dipeptide boronic acids but with slow recovery kinetics. That is, the enzyme can regain its enzymatic activity following exposure to the inhibitor but it does so slowly as compared to wild type proteins. These FAP alpha dimer enzyme forms can be used, inter alia, in combination with an inhibitor to control the level of enzymatic activity in a subject. For example, such forms may be provided in a pharmaceutically acceptable injectable preparation together with inhibitors, among them dipeptide proline boronic acids, at such a concentration and for such a period as necessary to allow binding ofthe inhibitor. Enzyme treated in this manner may be stored as necessary for intermediate periods under conditions conducive to the preservation ofthe inhibited complex, including freezing or cooling on ice or by other means, or by the manipulation of pH. Concentrations of approximately 0.5 nM inhibitor or higher but typically not greater than 10 μM and time periods approximately 1 -15 min or longer for complex formation are preferable but not limiting. Preferred dipeptide proline boronic acids include Val-boroPro and Ala-boroPro. The dipeptide proline boronic acids may also possess one or both amino acids in the unnatural D- configuration. Alterations to the nature ofthe first amino acid and the stereochemical configuration ofthe boronic acid to modulate the duration of inhibition and kinetics of release are contemplated embodiments. Other embodiments include mixing of inhibitors or of enzyme preparations treated with different inhibitors as a means of flattening out (i.e., plateauing) the released activity versus time profile from simple first order kinetics. Examples of mutants suitable for slow-release formulations include FAP A657D and DPPIV D663 A, among others. Also, heterodimers between different slow release mutant forms of FAP alpha dimer enzyme or with DPPIV is also envisioned.

Conditions to be treated: The invention provides methods to increase the level of FAP alpha dimer enzymatic activity in vivo. FAP alpha dimer enzymatic activity may be increased in vivo by administering FAP alpha dimer enzymes or nucleic acids encoding such proteins. FAP alpha dimer enzymes are used to down-regulate immune responses in vivo. In important embodiments, the immune responses are abnormal immune responses. An abnormal immune response is an immune response that is either inappropriate (e.g., is not functioning to eradicate an infection or other condition for which an immune response would be needed) or uncontrolled. An abnormal immune response in the context ofthe invention generally refers to hyperimmxinity. Examples include inflammation and inflammatory conditions, autoimmune disease, sepsis and septic shock (e.g., endotoxic shock), cytokine induced shock, allergies or bronchitis (including chronic allergies and chronic bronchitis), asthma, uncontrolled immune responses associated with particular infections such as RS V, graft versus host disease (GVHD), tissue, skin and organ transplantation rejection, osteoporosis, psoriasis, acute pancreatitis, premature labor secondary to intrauterine infections, chronic inflammatory pathologies with or without autoimmune involvement, fever and the like. Treatment of these conditions with FAP alpha can reduce symptoms or slow disease development. "Inflammation" is a localised protective response elicited by a foreign (non-self) antigen, and/or by an injury or destruction of tissue(s), which serves to destroy, dilute or sequester the foreign antigen, the injurious agent, and/or the injured tissue. Inflammation generally occurs when tissues are injured by viruses, bacteria, trauma, chemicals, heat, cold or any other harmful stimuli. In such instances, T cells, B cells and macrophages work with other cells and soluble products that are mediators of inflammatory responses including neutrophils, eosinophils, basophils, kinin and coagulation systems, and complement cascade. In another important embodiment, the inflammation is caused by an immune response against "self-antigen," and the subject in need of treatment according to the invention has an autoimmune disease. "Autoimmune disease" as used herein, results when a subject's immune system attacks its own organs or tissues, producing a clinical condition associated with the destruction of that tissue, as exemplified by diseases such as rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, autoimmune pulmonary inflammation, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre's syndrome, psoriasis, thyroiditis, Graves' disease, autoimmune thyroiditis, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus, autoimmune inflammatory eye disease, etc. Examples include chronic and acute inflammatory conditions such as but not limited to arthritis, rheumatoid arthritis, chronic inflammatory arthritis, inflammation associated with pulmonary disease, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), inflammation resulting from allergic reactions or acute or chronic infections (caused by viral, bacterial, fungal, protozoan or other organisms), systemic lupus erythematosus, atherosclerosis, airway inflammatory disease, tendonitis, inflammatory stage of alopecia, insect bites, multiple sclerosis, chronic inflammation in the brain and thrombotic disease, pulmonary fibrosis, psoriasis and hypersensitivity skin disease. Arthritis is a chronic joint disease characterized by the inflammation of synovial tissue and by a progressive degradation ofthe molecular components constituting the joint cartilage and bone. Inflammatory bowel disease ("IBD") refers to an acute or chronic inflammatory autoimmxme condition affecting the gastrointestinal tract and associated with one or more of the following symptoms: transmural acute and chronic granulomatous inflammation with ulceration, crypt abbesses, marked fibrosis, spontaneous reactivation, extraintestinal inflammation and anemia. IBD generally refers to two distinct conditions known as ulcerative colitis and Crohn's disease. Ulcerative colitis is a mucosal ulceration ofthe colon. Crohn's disease, also known as ileitis, ileocolitis and colitis, is a transmural inflammation that can be found throughout the general intestinal tract. Examples of non-human autoimmune conditions include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, insulin dependent diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis. The condition can also be the systemic response to diseases such as sepsis and pancreatitis. Sepsis is the systemic inflammatory response caused by microbial infection. Release of bacterial endotoxin from invading microbes stimulates the release of tumor necrosis factor alpha and IL-1, among other cytokines. Symptoms associated with sepsis include changes in thermoregulation, vascular permeability and resistance, cardiac function, bone marrow function, activity of key enzymes, drop in mean arterial blood pressure (MAP), decrease in cardiac output, tachycardia, tachypnea, lacticacidemia and leukopenia. Subjects at risk of developing sepsis, or that have developed sepsis are treated according to the invention. One example of a subject at risk of developing sepsis is a subject that will undergo surgery. Pancreatitis is acute or chronic inflammation ofthe pancreas, which may be asymptomatic or symptomatic and which is due to autodigestion ofthe pancreatic tissue by its own enzymes. It may be caused by alcoholism or biliary tract disease, hyperlipaemia, hyperparathyroidism, abdominal trauma, vasculitis or uraemia. The condition can be an allergic reaction and conditions associated therewith (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens- Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or bronchitis (including chronic bronchitis) and other respiratory problems. The allergic reaction may also be to chemical or biological substances such as penicillin. The method ofthe invention may also be used to induce a state of tolerance or anergy. These latter states may be appropriate during pregnancy (to prevent a mother from developing an immune response to her child, particularly with respect to the Rh antigen), in autoimmune conditions in which the body inappropriately identifies its own organs and cells are being foreign and thus mounts an immune response against them, and in organ transplantation to prevent organ rejection. Tolerance generally refers to a state in which T cells are rendered non-responsive or anergic. It may be demonstrated by the absence of a T cell response upon subsequent exposure to a particular antigen.

IL-1 mediated conditions: In some instances, FAP alpha dimer enzymes are administered to subjects having an IL-1 mediated condition. An IL-1 mediated condition as used herein is a disease or medical condition associated with elevated levels of IL-1 in bodily fluids or tissue. IL-1 is a cytokine that is produced as a result of infections and various kinds antigenic stimulation. IL-1 protein has a molecular weight of about 17.5 kDa in its mature form, and is produced primarily by the macrophages but also by epidermal, lymphoid, vascular and epithelial cells. IL-1 is a key cytokine in the body's ability to mount an inflammatory and immune response. It can however also act as a hormone, inducing metabolic, neurological, hematological and endocrinological changes. IL-1 exists in two active forms. The predominant form is IL-1 beta which is initially synthesized as an inactive precursor of 269 amino acids (31 kDa). This precursor is then cleaved to give rise to a mature form having amino acids 117-269 ofthe precursor form. The much less frequent form of IL-1 is IL-1 alpha which is about 26% homologous with IL-1 beta. It is initially synthesized as an active precursor form of 271 amino acids, which when cleaved gives rise to the mature form. IL-1 alpha and IL-1 beta are coded by distinct genes. IL-1 alpha and IL-1 beta recognize and bind to the same receptor on the cell surface (IL-1R). IL-1 beta is a cytokine that acts to increase the production of other cytokines and chemokines¹'⁴². This activity is described as proinflammatory, and can contribute to the pathology of inflammatory autoimmune diseases such as rheumatoid arthritis and type I diabetes . Inhibition of dipeptidyl peptidase activity in vivo by administration of PT-100 to mice induces both increased production of IL-1 beta and other cytokines and chemokines. The cytokine and chemokine responses to PT-100 require IL-1 beta signaling because, in mice which lack the IL-1 receptor, these chemokine responses were found to be greatly reduced (FIG. 3). Similarly to the in vitro response of bone marrow stromal cells, in vivo responses to PT-100 in mice appeared to be due to FAP inhibition because the induction of increased cytokine and chemokine production was undiminished in the absence of CD26 (FIG. 4). Overall, the data indicate that inhibition of FAP results in the stimulation of IL-1 beta production that in turn induces the expression of other cytokines and chemokines involved in immune and inflammatory responses. IL-1 has been shown to play a role in many conditions. Autoimmune or inflammatory diseases in which IL-1 is involved include rheumatoid arthritis³'⁴, insulin dependent diabetes (type I diabetes)⁵, septic shock⁶'⁷, inflammatory bowel disease², and atherosclerosis². In addition, a linkage between IL-1 and disease has been suggested in transplant rejection, graft- versus-host disease (GVHD), psoriasis, asthma, osteoporosis, osteoarthritis, periodontal disease, autoimmune thyroiditis, alcoholic hepatitis, premature labor secondary to uterine infection atherosclerosis, Guillain-Barre's syndrome and sleep disorders ' .

Co-administration: FAP alpha dimer enzymes can be administered to subjects either alone or in combination with other agents. As an example, FAP alpha dimer enzymes can be administered in combination with immunosuppressants, anti-inflammatory agents, anti- infectives, and the like. In some instances, the agents are administered substantially simultaneously with each other. By "substantially simultaneously," it is meant that the FAP alpha dimer enzyme is administered to a subject close enough in time with the administration of second (preferably therapeutic) agent, whereby the second agent may exert a potentiating effect on FAP alpha dimer enzyme activity. Thus, by substantially simultaneously it is meant that the FAP alpha dimer enzyme is administered before, at the same time, and/or after the administration ofthe second agent. As will be described below, FAP alpha dimer enzyme can be administered as a polypeptide, and/or a nucleic acid that encodes the polypeptide. In certain embodiments, the second agents are immunosuppressants. An immunosuppressant is an agent that down-regulates an immune response or prevents the initiation of an immune response. These include but are not limited to Azathioprine;

Azathioprine Sodium; Cyclosporine; Daltroban; Gusperimus Trihydrochloride; Sirolimus; and

Tacrolimus. In other embodiments, the second agents are anti-inflammatory agents. Anti- inflammatory agents are agents that prevent or down-regulate inflammation. These include but are not limited to Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha

Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra;

Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;

Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen;

Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;

Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;

Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl

Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac;

Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;

Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen;

Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol

Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen

Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;

Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;

Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone

Suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol;

Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate;

Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate;

Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium. Anti-infectives include anti-bacterial agents, anti-viral agents, anti-fungal agents, anti- parasitic agents, anti-mycobacterial agents and the like. Anti-bacterial agents kill or inhibit the growth or function of bacteria. A large class of anti-bacterial agents is antibiotics. Antibiotics, which are effective for killing or inhibiting a wide range of bacteria, are referred to as broad spectrum antibiotics. Other types of antibiotics are predominantly effective against the bacteria ofthe class gram-positive or gram- negative. These types of antibiotics are referred to as narrow spectrum antibiotics. Other antibiotics which are effective against a single organism or disease and not against other types of bacteria, are referred to as limited spectrum antibiotics. Anti-bacterial agents are sometimes classified based on their primary mode of action. In general, anti-bacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors. Cell wall synthesis inhibitors inhibit a step in the process of cell wall synthesis, and in general in the synthesis of bacterial peptidoglycan. Cell wall synthesis inhibitors include β-lactam antibiotics, natural penicillins, semi-synthetic penicillins, ampicillin, clavulanic acid, cephalolsporins, and bacitracin. The β-lactams are antibiotics containing a four-membered β-lactam ring which inhibits the last step of peptidoglycan synthesis, β-lactam antibiotics can be synthesized or natural. The natural antibiotics are generally produced by two groups of fungi, penicillium and cephalosporium molds. The β-lactam antibiotics produced by penicillium are the natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of penicillium chrysogenum. The natural penicillins have a narrow spectrum of activity and are generally effective against streptococcus, gonococcus, and staphylococcu . Other types of natural penicillins, which are also effective against gram-positive bacteria, include penicillins F, X, K, and O. Semi-synthetic penicillins are generally modifications ofthe molecule 6- aminopenicillanic acid produced by a mold. The 6-aminopenicillanic acid can be modified by addition of side chains which produce penicillins having broader spectrums of activity than natural penicillins or various other advantageous properties. Some types of semi-synthetic penicillins have broad spectrums against gram-positive and gram-negative bacteria, but are inactivated by penicillinase. These semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin. Other types of semi-synthetic penicillins have narrower activities against gram-positive bacteria, but have developed properties such that they are not inactivated by penicillinase. These include, for instance, methicillin, dicloxacillin, and nafcillin. Some ofthe broad spectrum semi-synthetic penicillins can be used in combination with β-lactamase inhibitors, such as clavulamic acids and sulbactam. The β-lactamase inhibitors do not have anti-microbial action but they function to inhibit penicillinase, thus protecting the semi-synthetic penicillin from degradation. Another type of β-lactam antibiotic is the cephalolsporins. Cephalolsporins are produced by cephalolsporium molds, and have a similar mode of action to penicillin. They are sensitive to degradation by bacterial β-lactamases, and thus, are not always effective alone. Cephalolsporins, however, are resistant to penicillinase. They are effective against a variety of gram-positive and gram-negative bacteria. Cephalolsporins include, but are not limited to, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, and moxalactam. Bacitracin is another class of antibiotics which inhibit cell wall synthesis. These antibiotics, produced by bacillus species, prevent cell wall growth by inhibiting the release of muropeptide subunits or peptidoglycan from the molecule that delivers the subunit to the outside ofthe membrane. Although bacitracin is effective against gram-positive bacteria, its use is limited in general to topical administration because of its high toxicity. Carbapenems are another broad spectrum β-lactam antibiotic, which is capable of inhibiting cell wall synthesis. Examples of carbapenems include, but are not limited to, imipenems. Monobactems are also broad spectrum β-lactam antibiotics, and include, euztreonam. An antibiotic produced by streptomyces, vancomycin, is also effective against gram-positive bacteria by inhibiting cell membrane synthesis. Another class of anti-bacterial agents is cell membrane inhibitors. These compounds disorganize the structure or inhibit the function of bacterial membranes. Alteration ofthe cytoplasmic membrane of bacteria results in leakage of cellular materials from the cell. Compounds that inhibit or interfere with the cell membrane cause death ofthe cell because the integrity ofthe cytoplasmic and outer membranes is vital to bacteria. One clinically useful anti-bacterial agent that is a cell membrane inhibitor is Polymyxin, produced by Bacillus polymyxis. Polymyxins interfere with membrane function by binding to membrane phospholipids. Polymyxin is effective mainly against Gram- negative bacteria and is generally used in severe Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. Other cell membrane inhibitors include Amphotericin B and Nystatin produced by the bacterium Streptomyces which are also anti-fungal agents, used predominantly in the treatment of systemic fungal infections and Candida yeast infections respectively.

Imidazoles, produced by the bacterium Streptomyces, are another class of antibiotic that is a cell membrane inhibitor. Imidazoles are used as anti-bacterial agents as well as anti-fungal agents, e.g., used for treatment of yeast infections, dermatophytic infections, and systemic fungal infections. Imidazoles include but are not limited to clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole. Many anti-bacterial agents are protein synthesis inhibitors. These compounds prevent bacteria from synthesizing structural proteins and enzymes and thus cause inhibition of bacterial cell growth or function or cell death. In general these compounds interfere with the processes of transcription or translation. Anti-bacterial agents that block transcription include but are not limited to Rifampins, produced by the bacterium Streptomyces and Ethambutol, a synthetic chemical. Rifampins, which inhibit the enzyme RNA polymerase, have a broad spectrum activity and are effective against gram-positive and gram-negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is effective against Mycobacterium tuberculosis. Anti-bacterial agents which block translation interfere with bacterial ribosomes to prevent mRNA from being translated into proteins. In general this class of compounds includes but is not limited to tetracyclines, chlorarnphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin). Some of these compounds bind irreversibly to the 30S ribosomal subunit and cause a , misreading ofthe mRNA, e.g., the aminoglycosides. The aminoglycosides are a class of antibiotics which are produced by the bacterium Streptomyces, such as, for instance streptomycin, kanamycin, tobramycin, amikacin, and gentamicin. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram- negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is used against many strains of Gram-positive and Gram- negative bacteria, including Pseudomonas infections, especially in combination with Tobramycin. Kanamycin is used against many Gram-positive bacteria, including penicillin- resistant staphylococci. Another type of translation inhibitor anti-bacterial agent is the tetracyclines. The tetracyclines bind reversibly to the 3 OS ribosomal subunit and interfere with the binding of charged tRNA to the bacterial ribosome. The tetracyclines are a class of antibiotics, produced by the bacterium Streptomyces, that are broad-spectrum and are effective against a variety of gram-positive and gram-negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline, and chlortetracycline. They are important for the treatment of many types of bacteria but are particularly important in the treatment of Lyme disease. Anti-bacterial agents such as the macrolides bind reversibly to the 50S ribosomal subunit and inhibits elongation ofthe protein by peptidyl transferase or prevents the release of uncharged tRNA from the bacterial ribosome or both. The macrolides contain large lactone rings linked through glycoside bonds with amino sugars. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enter obacteriaceae. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram-positive bacteria. Another type of translation inhibitor is chlorarnphenicol. Chlorarnphenicol binds the 70S ribosome inhibiting the bacterial enzyme peptidyl transferase thereby preventing the growth ofthe polypeptide chain during protein synthesis. Chlorarnphenicol can be prepared from Streptomyces or produced entirely by chemical synthesis. Some anti-bacterial agents disrupt nucleic acid synthesis or function, e.g., bind to DNA or RNA so that their messages cannot be read. These include but are not limited to quinolones and co-trimoxazole, both synthetic chemicals and rifamycins, a natural or semi- synthetic chemical. The quinolones block bacterial DNA replication by inhibiting the DNA gyrase, the enzyme needed by bacteria to produce their circular DNA. They are broad spectrum and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase) which is essential for DNA replication and allows supercoils to be relaxed and reformed, inhibiting DNA gyrase activity. The main use of nalidixic acid is in treatment of lower urinary tract infections (UTI) because it is effective against several types of Gram- negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of UTI. Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim, wliich blocks the bacterial synthesis of folic acid needed to make DNA nucleotides. Rifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis) and some Gram-negative bacteria. Rifampicin binds to the beta subunit ofthe polymerase and blocks the addition ofthe first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis. Another class of anti-bacterial agents is compounds that function as competitive inhibitors of bacterial enzymes. The competitive inhibitors are mostly all structurally similar to a bacterial growth factor and compete for binding but do not perform the metabolic function in the cell. These compounds include sulfonamides and chemically modified forms of sulfanilamide which have even higher and broader anti-bacterial activity. The sulfonamides (e.g. gantrisin and trimethoprim) are useful for the treatment of Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli, and have been used in the treatment of uncomplicated UTI caused by Ε. coli, and in the treatment of meningococcal meningitis. Anti-viral agents are compounds which prevent infection of cells by viruses or replication ofthe virus within the cell. There are several stages within the process of viral infection which can be blocked or inhibited by antiviral agents. These stages include, attachment ofthe virus to the host cell (immunoglobulin or binding peptides), uncoating of the virus (e.g. amantadine), synthesis or translation of viral mRNA (e.g. interferon), replication of viral RNA or DNA (e.g. nucleoside analogues), maturation of new virus proteins (e.g. protease inhibitors), and budding and release ofthe virus. Nucleotide analogues are synthetic compounds which are similar to nucleotides, but which have an incomplete or abnormal deoxyribose or ribose group. Once the nucleotide analogues are in the cell, they are phosphorylated, producing the triphosphate formed which competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form ofthe nucleotide analogue is incorporated into the growing nucleic acid chain, it causes irreversible association with the viral polymerase and thus chain termination. Nucleotide analogues include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), gancyclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of respiratory syncitial virus), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine). Anti-fungal agents are useful for the treatment and prevention of infective fungi.

Anti-fungal agents are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase. These include, but are not limited to, basiungin/ECB. Other anti-fungal agents function by destabilizing membrane integrity. These include, but are not limited to, imidazoles, such as clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine, and turbinifine. Other anti-fungal agents function by breaking down chitin (e.g. chitinase) or immunosuppression (501 cream). Still other anti-fungal agents include prednisone, disodium chromoglycat, nystatin, hydroxystilbamidine, 5-fluorocytosine, pimaricin, turbinifine, gentian violet, resorcin, iodine, thiabendazole, glutarardehyde, tolnaftate, econazole, sulfonamides, phyfluorocytozine, and oral potassium iodide. Parasiticides are agents that kill parasites directly. Such compounds are known in the art and are generally commercially available. Examples of parasiticides useful for human administration include but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide some of which are used alone or in combination with others. Parasiticides used in non-human subjects include piperazine, diethylcarbamazine, thiabendazole, fenbendazole, albendazole, oxfendazole, oxibendazole, febantel, levamisole, pyrantel tartrate, pyrantel pamoate, dichlorvos, ivermectin, doramectic, milbemycin oxime, iprinomectin, moxidectin, N-butyl chloride, toluene, hygromycin B thiacetarsemide sodium, melarsomine, praziquantel, epsiprantel, benzimidazoles such as fenbendazole, albendazole, oxfendazole, clorsulon, albendazole, amprolium; decoquinate, lasalocid, monensin sulfadimethoxine; sulfamethazine, sulfaquinoxaline, metronidazole. Parasiticides used in horses include mebendazole, oxfendazole, febantel, pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; for S. westeri: ivermectin, benzimiddazoles such as thiabendazole, cambendazole, oxibendazole and fenbendazole. Useful parasiticides in dogs include milbemycin oxine, ivermectin, pyrantel pamoate and the combination of ivermectin and pyrantel. The treatment of parasites in swine can include the use of levamisole, piperazine, pyrantel, thiabendazole, dichlorvos and fenbendazole. In sheep and goats anthelmintic agents include levamisole or ivermectin. Caparsolate has shown some efficacy in the treatment of D. immitis (heartworm) in cats. Agents used in the prevention and treatment of protozoal diseases in poultry, particularly trichomoniasis, can be administered in the feed or in the drinking water and include protozoacides such as aminonitrothiazole, dimetridazole (Emtryl), nithiazide

(Hepzide) and Enheptin. However, some of these drugs are no longer available for use in agricultural stocks in the USA.

Delivery of FAP alpha dimer enzyme: As stated above, in some instances, soluble FAP is administered as a nucleic acid or a protein. In some embodiments, the nucleic acids or proteins/peptides are isolated. In still further embodiments, the nucleic acids or proteins/peptides are substantially pure. As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage ofthe material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins/peptides, the term "isolated" means separated from its native environment in sufficiently pure form so that it can be manipulated or used for any one ofthe purposes ofthe invention. Thus, isolated means sufficiently pure to be used (i) to raise and/or isolate antibodies, (ii) as a reagent in an assay, or (iii) for sequencing, etc. The term "substantially pure" means that the nucleic acid or protein/peptide is essentially free of other substances with which it may be found in nature or in vitro systems, to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be produced by techniques well known in the art. As an example, because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight ofthe preparation. The protein is nonetheless isolated in that it has been separated from many ofthe substances with which it may be associated in living systems, i.e. isolated from certain other proteins. The invention embraces the use of nucleic acids that encode the FAP alpha dimer enzymes described herein, including degenerates, homologs and alleles thereof. Homologs and alleles ofthe FAP nucleic acids can be identified by conventional techniques. Thus, an aspect ofthe invention is those nucleic acid sequences which code for FAP alpha dimer enzyme and which hybridize to a nucleic acid molecule consisting ofthe coding region of SEQ ID NO: 1 (e.g., nucleotides 209 to 2488), under stringent conditions. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH₂PO₄(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2x SSC at room temperature and then at O.lx SSC/0.1% SDS at temperatures up to 68°C. There are other conditions, reagents, and so forth which can be used, and would result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of FAP alpha dimer enzyme nucleic acids ofthe invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing. In general homologs and alleles typically will share at least 75% nucleotide identity to SEQ ID NO: 3 (nucleotide sequence ofthe FAP portion used to make the soluble FAP and the nucleotide sequence of soluble FAP itself), and/or at least 90% amino acid identity to SEQ ID NO: 4 (amino acid sequence ofthe FAP portion used to make the soluble FAP and the amino acid sequence of soluble FAP itself) or SEQ ID NOs: 61 or 70. Preferably, homologs and alleles will share at least 85% nucleotide identity and/or at least 95% amino acid identity and, even more preferably, at least 95% nucleotide identity and/or at least 99% amino acid identity will be shared. The homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the NCBI website on the internet. Exemplary software tools include the BLAST system (see NIH website) using default settings. Pairwise and ClustalW alignments (BLOSUM30 and/or BLOSUM62 matrix settings) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVetor sequence analysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acids also are embraced by the invention. The invention also includes degenerate nucleic acids which include alternative codons to those present in FAP alpha dimer enzyme nucleic acids provided herein. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any ofthe serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating FAP alpha dimer enzyme. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy ofthe genetic code. The invention also contemplates mutations to the nucleic acids encoding FAP alpha dimer enzyme that are silent as to the amino acid sequence ofthe protein, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a FAP alpha dimer enzyme nucleic acid or cDNA clone to enhance expression ofthe polypeptide. The methods ofthe invention may also utilize vectors containing the nucleic acid for FAP alpha dimer enzyme, and cells transfected with such vectors. Virtually any cells, prokaryotic or eukaryotic, which can be transformed with heterologous DNA or RNA and which can be grown or maintained in culture or which can be introduced into a subject, may be used in the practice ofthe invention. Examples include bacterial cells such as E. coli, insect cells, and mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, including mast cells, fibroblasts, oocytes and lymphocytes, and they may be primary cells or cell lines. Specific examples include CHO cells and COS cells. Cell-free transcription systems also may be used in lieu of cells. As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication ofthe desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., beta-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which they are operably joined. The FAP alpha dimer enzyme nucleic acid would commonly be placed under the control of a regulatory sequence. Regulatory sequences include, but are not limited to, promoters, and other elements which although capable of affecting transcriptional levels are not, in and of themselves, sufficient for such transcription. Examples of these latter elements include enhancers and repressor elements. Minimal promoter elements have been recognized in the art and include sequences such as a CCAAT box or a TATA sequence. Suitable marker sequences for these purposes are similar to those described above. As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription ofthe coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription ofthe coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability ofthe corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. The precise nature ofthe regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CCAAT sequence, and the like. Especially, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control ofthe operably joined coding sequence. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors ofthe invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous nucleic acid, usually DNA, molecules, encoding a soluble FAP polypeptide or fragment or a variant thereof. The heterologous nucleic acid molecules are placed under operable control of transcriptional elements to permit the expression ofthe heterologous nucleic acid molecules in the host cell. Preferred systems for mRNA expression in mammalian cells are those such as pcDNA3.1 (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen,

Carlsbad, CA), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor 1- alpha, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992). The use ofthe adenovirus as an Adeno.Pl A recombinant is disclosed by Warmer et al., in intradermal injection in mice for immunization against P1A (Int. J. Cancer, 67:303-310, 1996). An example of a commercially available secretion vector for mammalian expression is pSecTag2B vector (InVitrogen Corporation). Generally, the FAP alpha dimer enzyme amino acid sequence to be expressed from these vectors should be fused to a signal sequence in order to ensure release of FAP alpha dimer enzyme. The invention embraces the use ofthe above described, FAP alpha dimer enzyme nucleotide sequence containing expression vectors, to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as human, mouse, hamster, pig, goat, primate, etc., from a wide variety of tissue types including primary cells and established cell lines. Specific examples include mammalian epithelial cells, fibroblast cells and kidney epithelial cells, either as primary cells or cell lines. Production of recombinant FAP alpha dimer enzyme heterodimers is accomplished by transfection ofthe chosen cell line with a two or more plasmids which encode different forms of FAP alpha dimer enzyme, or a mixture of FAP and DPPIV expressing plasmids in proportions deemed optimal for the desired outcome. In some instances, it may be desirable to coat or load FAP alpha dimer enzyme onto material surfaces. "Material surfaces" as used herein, include, but are not limited to, dental and orthopedic prosthetic implants, artificial valves, and organic implantable tissue such as a stent, allogeneic and/or xenogeneic tissue, organ and/or vasculature. Implantable prosthetic devices have been used in the surgical repair or replacement of internal tissue for many years. Orthopedic implants include a wide variety of devices, each suited to fulfill particular medical needs. Examples of such devices are hip joint replacement devices, knee joint replacement devices, shoulder joint replacement devices, and pins, braces and plates used to set fractured bones. Some contemporary orthopedic and dental implants, use high performance metals such as cobalt-chrome and titanium alloy to achieve high strength. These materials are readily fabricated into the complex shapes typical of these devices using mature metal working techniques including casting and machining. In important embodiments, the material surface is part of an implant. The material surface is coated with an amount of FAP alpha dimer enzyme effective to down-regulate an abnormal immune response in the vicinity ofthe material surface. This may entail preventing the migration, accumulation or activation of immune cells in the vicinity of the material surface.

A subject shall mean a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjects suitable for therapeutic or prophylactic methods include vertebrate and invertebrate species. Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals

(e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many ofthe embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates. The compositions, as described above, are administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome. It will also depend upon, as discussed above, the stage ofthe condition, the age and physical condition ofthe subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result. In some cases an effective amount is that amount that down-regulates an immune response. Down-regulation of an immune response can be assessed in a number of ways. These include measuring white blood cell counts either locally or systemically (including neutrophil, macrophage and T cell counts), body temperature ofthe subject (e.g., presence or absence of a bodily temperature over 37.5°C, levels of cytokines or immunomodulators in a subject, swelling, pain, joint flexibility, range of motion, and the like. In some cases, IL-1 levels in a subject may be measured as an indicator of immune response down-modulation. Effective amounts may reduce IL-1 levels to a normal level or to a below normal level. A normal level of IL-1 is the level of IL-1 in a subject that is not experiencing an IL-1 mediated condition or any other condition that would impact upon IL-1 levels. Normal IL-1 levels in human serum are less than 4 pg/ml for both IL-1 alpha and IL-1 beta, individually. Cell and cytokine or mediator levels may be measured in a bodily fluid from a subject including but not limited to blood, serum, plasma and cerebrospinal fluid. Generally, doses of active compounds ofthe present invention would be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses ranging from 50-500 mg/kg will be suitable. The methods ofthe invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels ofthe active compounds without causing clinically unacceptable adverse effects. A variety of administration routes are available including but not limited to oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations, such as for example in a sepsis situation. Generally, administration by injection is preferred. When peptides are used therapeutically, in certain embodiments one desirable route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing peptides are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties ofthe antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing protein or peptide aerosols without resort to undue experimentation. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous veliicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-infectives, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. The agents may be combined, optionally, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components ofthe pharmaceutical compositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The invention in other aspects includes pharmaceutical compositions ofthe agents. When administered, the pharmaceutical preparations ofthe invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptably compositions. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope ofthe invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Various techniques may be employed for introducing nucleic acids ofthe invention into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid-CaPO₄ precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid to particular cells. In such instances, a vehicle used for delivering a nucleic acid ofthe invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle. For example, where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids. Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations ofthe FAP alpha dimer enzyme, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules ofthe foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. A preferred delivery system ofthe invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system ofthe invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2-4.0 μm can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci, (1981) 6:77). In order for a liposome to be an efficient gene transfer vector, one or more ofthe following characteristics should be present: ( 1 ) encapsulation of the gene of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery ofthe aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information. Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis, G. in Trends in Biotechnology, (1985) 3:235-241. In one important embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled

"Polymeric Gene Delivery System"). PCT/US/03307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instant invention, the fugetactic agents described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein an agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein an agent is stored in the core of a polymeric shell). Other forms ofthe polymeric matrix for containing an agent include films, coatings, gels, implants, and stents. The size and composition ofthe polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method of delivery which is to be used. Preferably when an aerosol route is used the polymeric matrix and agent are encompassed in a surfactant vehicle. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. In another important embodiment the delivery system is a biocompatible microsphere that is suitable for local, site-specific delivery. Such microspheres are disclosed in Chickering et al., Biotech. AndBioeng, (1996) 52:96-101 and Mathiowitz et al, Nature, (1997) 386:.410- 414. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents ofthe invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers. In general, agents are delivered using a bioerodible implant by way of diffusion, or more preferably, by degradation ofthe polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene, polyvinylpyrrolidone, and polymers of lactic acid and gly colic acid, polyanhydrides, poly(ortho)esters, poly(butiric acid), poly(valeric acid), and poly(lactide- cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). In addition, important embodiments ofthe invention include pump-based hardware delivery systems, some of which are adapted for implantation. Such implantable pumps include controlled-release microchips. A preferred controlled-release microchip is described in Santini, JT Jr., et al., Nature, 1999, 397:335-338, the contents of which are expressly incorporated herein by reference. Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, as used herein, means that the implant is constructed and arranged to delivery therapeutic levels ofthe active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some ofthe release systems described above. As discussed above, in certain embodiments, the agents ofthe invention are delivered directly to the site at which there is inflammation, e.g., the joints in the case of a subject with rheumatoid arthritis, the blood vessels of an atherosclerotic organ, etc. For example, this can be accomplished by attaching an agent (nucleic acid or polypeptide) to the surface of a balloon catheter; inserting the catheter into the subject until the balloon portion is located at the site of inflammation, e.g. an atherosclerotic vessel, and inflating the balloon to contact the balloon surface with the vessel wall at the site ofthe occlusion. In this manner, the compositions can be targeted locally to particular inflammatory sites to modulate immune cell migration to these sites. In another example the local administration involves an implantable pump to the site in need of such treatment. Preferred pumps are as described above. In a further example, when the treatment of an abscess is involved, the agent may be delivered topically, e.g., in an ointment/dermal formulation. Optionally, the agents are delivered in combination with other therapeutic agents (e.g., anti-inflammatory agents, immunosuppressant agents, etc.).

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments ofthe invention and are not to be construed to limit the scope ofthe invention. Examples

Abbreviations: The following abbreviations are used throughout the specification and claims and in the Examples:

"a.a.": amino acid.

Amino acid single letter code: A=Alanine; C=cysteine; D=aspartic acid; E=glutamic acid; F=phenylalanine; G=glycine; H=histidine; I=isoleucine; K=lysine; L=leucine; M=methionine; N=asparagines; P=proline; Q=glutamine; R=arginine; S=serine; T=threonine; N=valine; W=tryptophan; Y=tyrosine.

Amino acid mutations: e.g. A657D denoted alanine residue 657 mutated to aspartate. "Nitrilo- derivative": A class of Xaa-proline dipeptide inhibitors characterized by replacement ofthe carboxyl group ofthe proline with the nitrilo (cyano) group. Xaa = any amino acid.

"Overlap extension PCR": Overlap extension PCR is a polymerase chain reaction (PCR) based technique for insertion of mutations or restriction sites at any point in a DNA molecule (Kadowaki et al. 1989. Gene. 76, 161-166). It proceeds in two successive rounds of PCR. Typically, in the first round, two separate PCR reactions (Tube A and Tube B) are run. In each, one of a pair of divergent overlapping mutagenic primers is paired with an external non- mutagenic primer to give overlapping PCR products with the mutation at their 3 ' in one case and 5' end in the other. The mutagenic primers are designed to ensure productive annealing to the template on one hand, typically with 6-20 perfectly matched nucleotides 3' ofthe mutation(s), and with each other on the other. The two PCR products from the first round are then mixed and PCR repeated with only the external primers. The overlap near the mutagenic site allows the products to anneal and so prime each other, so that after fill-in by the thermostable polymerase, they become a single long product. The latter is then amplified in the same reaction by the external primers giving a product that can then be cut using available restriction sites. These flanking restriction sites can be any reasonable distance away from the site of mutation and their availability determines the location ofthe external primers. The resultant fragment is then used to replace the corresponding wild-type fragment, yielding the desired mutation.

"Primer": An oligonucleotide capable of annealing to a specified DNA target and serving as a priming site for DNA polymerase activity.

'RT-PCR". Polymerase chain reaction on cDNA derived from RNA via reverse transcription.

'sr hFAP": soluble recombinant human FAP

"sr hDPPIV": soluble recombinant human DPPIV.

Nal-boroPro": L-valine-pyrollidine-2-boronic acid dipeptide inhibitor. Example 1.1: Normal B6 mice (+/+) and congenic B6.129/s7-lllrltmllmx mice with a targeted mutation ofthe IL-1 receptor-1 (-/-) were orally administered 160-μg PT-100 or saline. Eight 5 hours after PT-100 administration, the levels of cytoldnes and chemokines indicated on the ordinates were determined by ELISA of serum or spleen protein extracts for IL-1 beta (FIG. 3). Data represent the increases observed in PT-100 treated mice after correction for control levels in saline-treated mice. IL-1 beta levels were normalized so as to correct for differences between the total protein concentrations in extracts. The data indicates that loss ofthe IL-10 beta receptor results in loss of production of other cytokines except IL-1 beta itself.

Example 1.2: BALB/c CD2tf wild-type and BALB/c CD26^"A mice were treated with various doses of PT-100 and the resulting cytokine and chemokine profile of these mice was5 analyzed. Dose response curves for the indicated cytokine or chemokine are shown in FIG. 4. Data represent the mean ±SE n=5) responses determined by ELISA of serum samples. CD26 knockout mice have a strong cytokine response to PT100 despite the loss of CD26, indicating that another dipeptidyl peptidase, likely FAP, is responsible for the observed response. 0 Example 1.3: Stromal cells from humans and Fischer D^" (CD26 mutant) rats were isolated and treated in vitro with PT-100. Stromal cells were incubated in multi well plates with or without the addition of PT-100 for 4 (human cells) or 8 (rat cells) hours. IL-1 beta levels in supernatants were determined by ELISA. Data represent the means of duplicate cultures for !5 each experiment. The levels of IL-1 beta in tissue culture supernatants of these cells after several hours of incubation are increased as shown in Table 1, indicating that CD26 is not essential to the IL-1 response induced by PT-100 administration.

Table 1. Stimulation of IL-1 beta production by PT-100 in human bone marrow stromal 0 cell cultures Source of stromal Experiment . _cejl_{s 0} IL-1 beta concentration (pg/ml) after incubation with : Medium 10 μM PT-100 Human 1 0.0 201.0 2 0.0 36.0 3 1.2 19.2 Fischer D^" rat 4 0.0 ⁷⁹-°

Example 1.4: Production of soluble recombinant human FAP: This strategy is based on information on the N-terminus of serum DPPIV (Durinx et al. Eur J Biochem. 2000 Sep;267(l 7):5608-l 3). A truncated FAP was engineered in which a signal/leader sequence was joined to the residue in FAP analogous to the N-terminus of serum DPPIV to allow secretion. The cDNA encoding the desired truncated human FAP alpha dimer enzyme is engineered into the mammalian secretion vector pSecTag2 (Cat. # V900-20, InVitrogen Corporation). The vector, available in A, B or C versions, representing three possible phases for gene fusion, contains an immunoglobulin-kappa light chain secretion signal followed by a selection of restriction sites for gene insertion. The fusion requires engineering a restriction site upstream ofthe chosen fusion amino acid in the 5' end ofthe FAP alpha dimer enzyme nucleic acid in phase with the chosen restriction site (Sfi I) in the vector secretion sequence. The chosen fusion amino acid in the 5' end ofthe FAP (Thr38) is 3' ofthe trans-membrane anchoring domain. The pSecTag2 version B and its Sfi I restriction site are chosen for the fusion because it minimizes the additional N-terminal, vector-encoded residues in the mature secreted protein. Construction ofthe fusion is in 3 stages. First, human FAP alpha dimer enzyme cDNA corresponding to nucleotide 161-2526 approximately of wild type FAP alpha dimer enzyme (GenBank Accession number NM_004460) is obtained by reverse transciptase then Taq DNA polymerase mediated PCR (RT-PCR) on RNA from human stromal cells grown from bone marrow, and inserted into vector pPCR2.1 (InVitrogen Corporation) using the T/A cloning method, giving a plasmid pTAhFAP#2. The primers for this are hFAPl (5' ccacgctctg aagacagaat tagc 3' SEQ ID NO: 11) and hFAP2 (5' tcagattctg atagaggctt gc 3' SEQ ID NO: 12). Next, the cloned FAP cDNA, characterized as containing a single point mutation T229M, is excised with flanking BamHI and Not I restriction enzymes (contributed by the cloning vector), and inserted into similarly-cut pSecTag2-B vector, and which gives the correct orientation relative to the secretion signal but retains the complete FAP coding sequence plus untranslated upstream sequence (plasmid #13). Completion ofthe final plasmid requires deletion ofthe first 37 amino acids of FAP and insertion of Sfi I restriction site upstream ofthe Thr38 codon to allow an in-phase junction to the Ig-kappa secretion sequence at the Sfi I site. The 5' PCR primer sequence for insertion ofthe Sfi I site is chosen so that the same primer served for both human and mouse constructs. This makes residue #40 a lysine, as found in mouse wild type FAP alpha dimer enzyme. It is to be understood that the invention also embraces soluble hFAP having a methionine at residue #40 (as in wild type hFAP). This 5' primer, named Sfi-FAP-B, had the sequence 5' GTAGTCGGCC CAGCCGGCCA CAAAGAGAGC TCTTACCCTG AAGGATATTT TAAATG 3', SEQ ID NO: 13 (Sfi I site underlined). PCR of FAP cDNA with this primer and a reverse primer located 3' ofthe unique Xba I site (located in the codons for amino acid 113-115), gives a PCR product of approx. 700 nt. The Sfi I-Xba I double digest on plasmid #18 is used to remove the native 5 ' end of FAP up to the internal Xba I site at codon 114 and to cut the FAP PCR product. The appropriate fragments of >5 kb and approx. 259 nt respectively are isolated from an agarose gel using standard procedures (known to those skilled in the art) and ligated to each other. After transformation into bacteria and screening of colonies, those with correct properties are sequenced to ensure the correct fusion junction and absence of PCR-induced mutations, giving plasmid #122 which is designated wild-type FAP. The N-terminus ofthe final mature amino acid sequence of cleaved secreted product will contain 6 amino acids from the vector, DAAQPA, SEQ ID NO: 14 , fused to the truncated FAP sequence, of which the first 13 amino acids are TKRALTLKDILNG, SEQ ID NO: 15 . FIG. 10 demonstrates soluble FAP and DPPIV activity in several harvests of tissue culture supernatant from plasmids #122 and #135 respectively. Two amounts of plasmid (10 micrograms and 20 micrograms) were used to transfect HEK293T cells in a 10 cm diameter dish and harvests taken at 23.5, 39.5, 51.5 and 62h after addition of DNA to the cells. Assays contained 100 microlitre 50mM HEPES/NaOH buffer pH 8.1, lmM (#122) or 0.1 mM (#135) Ala-Pro-AFC substrate and 11 microlitres tissue culture supernatant. Assays were incubated at 37C and stopped after 1.75 h with 100 microlitres 1M sodium acetate pH4.5 and fluorescence read at 505 nm (Excitation at 400 nm). Published sequence of N-terminus of serum hDPPIV and inferred analogous site for FAP:

hDPPIV: MKTPWKVLLGLLGAAALVTIITVPWLLNKGTDDATADSRKTYTLTDYLKN— (SEQ ID NO: 16) Serum DPP4*: SRKTYTLTDYLKN— (SEQ ID NO: 17) RKTYTLTDYLKN— (SEQ ID NO: 18) :.. I 1 I I .: hFAP: MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNG-- (SEQ ID NO 19)

Proposed N-terminus: TMRAL LKDILNG— (SEQ ID NO 20) iriFAP: MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNG— (SEQ ID NO 21) Proposed N-terminus: TKRALTLKDILNG— (SEQ ID NO 22)

pSecTag2 vector (InVitrogen):

S32 6T& GTG CTG CTC TGG GTT CCA QT TCC ACT GGT GAC £CG C-CC CAGCCG Val Leu Leu Leu Trp Val Pro Qly S« r Ihr <Sl y*Asrp Signal ctesvaiξe fcite (SEQ ID NO: 23 for nucleotide sequence, and SEQ ID NO: 24 for amino acid sequence)

DNA sequence of vector-FAP junction showing the 6 vector-derived amino acids included in the mature secreted protein: Sfil

5 ' ggt /gacgcggcccagccggcc ACAAAGAGAGCTCTTACCcTGAAGGATATTTTAZ ATG3 ' ( SEQ ID NO : 65 ) g /D A Q P A -T K R A L T L K D I L N G ( SEQ ID NO : 25 ) / Vector I FAP >

Example 1.5: Preparation of soluble FAP alpha dimer enzyme: DNA ofthe FAP alpha dimer enzyme containing plasmid is prepared on a approximately 400 μg scale from overnight 30 ml cultures in Luria broth with 100 μg ampicillin per ml using a commercial kit (Qiagen Maxiprep Kit). Ten (10) μg of DNA and 30 μl of Lipofectamine 2000 transfection reagent (InVitrogen Corporation) are used to transiently transfect 293T cells in 10 cm diameter tissue culture plates using the manufacturer's protocol. Cells are at greater than about 70% confluent in Freestyle 293 Expression Medium (InVitrogen Corporation) containing 2.5% fetal calf serum and standard antibiotics penicillin and streptomycin. Antibiotic-free medium is used for the initial 18-24 h of transfection, after which serum-free medium with antibiotics is employed. Culture supernatant containing the secreted recombinant enzyme is harvested 6-18 h later and again 24 h after addition of fresh serum-free medium and is stored in a cold room. FIG. 9 and FIG. 10 show FAP activity in several harvests of tissue culture supernatant from various plasmids expressing secreted soluble FAP alpha dimer enzyme.

Example 1.6: Assay of soluble FAP alpha dimer enzyme: A typical activity assay consists of 135 μl 50 mM HEPES/Na buffer pH 8.1 (or other pH), 140 mM NaCI, 10-15 μl enzyme-containing culture supernatant, dipeptide substrate Ala- Pro-(7-amino-4-trifluoromethyl coumarin) (abbreviated Ala-Pro-AFC) at typically 0.25-1 mM (unless Km determinations require variation) added from a 100 or 400 mM stock in dimethyl formamide. Other buffers can be substituted. Assays lacking either substrate or enzyme are set up in 96-well microtitre plates, pre-warmed at the desired incubation temperature between room temperature (22°C) to 37°C. Then the missing component is added to start the reaction and incubation continued at the desired temperature. Production ofthe fluorescent AFC product is either monitored continuously in a thermostatted fluorometer or after termination with one to one-tenth volume 1 M sodium acetate pH 4.5.

Example 1.7: Immunoprecipitation results with FAP-specific mAb: Soluble FAP alpha dimer enzyme was isolated from the supernatant according to two methods: capture on Protein G beads and capture on Protein G coated 96-well plates. These approaches are discussed below. The protocol for capture on Protein G beads is as follows: Tris buffer / NaCI / 1 % triton 100 μl Anti-FAP mAb supernatant 100 μl Soluble FAP supernatant 300 μl-1 ml Supernatant containing FAP alpha dimer enzyme was incubated with anti-FAP mAb (i.e., tissue culture medium from F19 anti-hFAP hybridoma) for 20 min on ice. Then 38 μl Protein G beads (50% v/v) were added and tubes rocked 1 hr at 4°C. Beads were washed 2X with Triton-containing buffer, then once with 600 μl 50 mM HEPES pH 8.1, 140 mM NaCI. Finally, beads were suspended in 500 ul 50 mM HEPES pH 8.1, 140 mM NaCI containing 100 μM Ala-Pro-AFC substrate, incubated at 37°C in a rocker for 10 min to 2.5 h, stopped with 0.1-1 vol 1 M NaOAc pH 4.5 and centrifuged to pellet beads. The protocol for capture on Protein G coated 96 well plates is as follows: 100 μl goat anti-mouse IgG (H+L) polyclonal antibody was captured in wells of Protein G coated 96 well plates (PIERCE Biotechnology) by incubating at room temperature for 1 h at room temp. Wells were then washed and to them was added 100 μl anti-hFAP hybridoma supernatant for 1 h at room temp. Unbound mAb was washed away and 100 μl cell extract / 293T supernatant containing FAP alpha dimer enzyme was added. The wells were incubated 1 h at 4°C, and then washed twice with Triton-containing buffer, and once with 50 mM HEPES pH 8.1 , 140 mM NaCI. To the wells were added 90 μl of 50 mM

HEPES pH 8.1, 140 mM NaCI were added, followed by addition of inhibitor PT-100 (10 μl of 10X), if needed, and incubation at room temp for 15-20 min, if inhibitor was used. Then, 10 μl Ala-Pro-AFC substrate (1 lx) was added giving 100 μM final concentration. Plates were incubated at 37°C for 40 min, stopped with 1 vol 1 M NaOAc pH 4.5 and the fluorescence read at 505 nm with excitation at 400 nm in a Molecular Dynamics Spectra Max GeminiXS Fluorescence plate reader. Activity of immunoprecipitated recombinant soluble human FAP is shown in FIGs. 6 and 8.

Example 1.8: Inhibition of soluble recombinant human FAP by PT-100: Assays are done in a dark-sided 96-well plate. PT100 stock (0.1 M in 0.1 M HCl) was thawed, diluted in assay buffer (50 mM HEPES pH 8.1, 140 mM NaCI) immediately before use and added to enzyme. The reaction conditions were as follows: 50 mM HEPES pH 8.1, 140 mM NaCI 160 ul Soluble FAP (culture supernatant) 10-20 ul Inhibitor (diluted to 20X) 10 ul

The enzyme was incubated for 10-20 min at room temp with the PT-100 in order to provide sufficient time for PT-100 to bind. The solution was then warmed 5-10 min at 37°C, following with 20 μl of 2.5 mM or 1 mM Ala-Pro-AFC substrate (10 mM stock is diluted in DMF by 10X to give final concentration of 0.1 mM) was added. The solution was incubated at 37°C for 20 min tol h. The reaction was stopped with 0.1-1 vol of 1 M NaOAc pH 4.5. Fluorescence was read at 505 nm with excitation at 400 nm in a Molecular Dynamics Spectra Max GeminiXS Fluorescence plate reader. Wild type FAP alpha dimer enzyme was immunoprecipitated from Triton XI 00 extracts of RPMI-7951 cells and soluble FAP alpha dimer enzyme was immunoprecipitated from supernatant of transiently transfected 293T cells using Protein G coated 96 well plates as described above. Assays in the presence of PT-100 were done as described above. The inhibitor assay was performed as described above. The results of this assay are shown in FIG. 8.

Conclusions: Human and mouse FAP can be produced in a recombinant soluble secreted form, as shown in FIGs. 5, 9 and 10. The recombinant soluble human FAP however is recognized by mouse-derived hFAP specific mAb F19, while the murine version is not. This indicates that the cloning procedure did not affect the epitope structure of either FAP form, as relates to the mAb F19. This is shown in FIG. 6. PT-100 inhibits recombinant hFAP in 293 T supernatants with IC₅o -20 nM, as shown in FIG. 7. There is no difference in IC₅₀ between immunocaptured FAP from a native source or produced from the recombinant plasmid, as shown in FIG. 8.

Example 2: Expression and secretion of soluble recombinant human FAP alpha dimer enzyme starting at codon #38 using a secretion sequence derived from the cytokine G- CSF: A plasmid is constructed containing a portion of wild type FAP alpha dimer enzyme fused in phase to a functional human G-CSF leader sequence using the overlap extension PCR technique. Round 1 , Tube A: G-CSF leader is obtained by PCR from cDNA from 15-24 h LPS-treated human bone marrow stromal cells using primers hG-CSF F (5' CCAAGCTG GCTAGC CACCATG gctggac ctgccacccagag, SEQ ID NO: 26) and hG-CSF leader-R (5' GGC TTC CTG CAC TGT CCA GAG TGC ACT 3', SEQ ID NO: 27). Round 1 Tube B: The human FAP 5' end is amplified with primers hG-CSF_FAP-F (5' GCACTCTGGA C AGTGCAGGA AGCC ACAAAG AGAGCTCTTA CCcTGAAGGA TATTTTA 3 ' , SEQ ID NO: 28) and any primer 3' ofthe Xbal site such as hFAP-Clal-R (5' GCA GGG TAA GTG GTA TCG ATA ATA AAT ATC CG 3', SEQ ID NO: 29). Round 2 mixes the 2 Round 1 PCR products with the flanking hG-CSF-F and hFAP-Clal-R primers, followed by Nhel and Xbal (or Clal) digestion and replacement ofthe corresponding piece in plasmid #122, #13 or #23. (See Examples 1.4 and 5.3 for origins of these numbered plasmids.)

Example 3: Expression and secretion of soluble recombinant human DPPIV containing a 6 amino acid N-terminal extension in a mammalian cell line: Total RNA is isolated from the Caco-2 colorectal carcinoma cell line (ATCC HTB-37) by standard Trizol/phenol/chloroform methodology. The purified RNA (approx. 2.5 μg in a 20 μl reaction) is used to make cDNA using oligo-dT primer and a commercial reverse Transcriptase (RT) kit (InVitrogen). An aliquot (2 microlitre) ofthe RT reaction is used to PCR amplify a truncated coding region of DPPIV starting at S39 with primers Sfi-DPPIV (5' GTAGTCGGCC CAGCCGGCC AGTCGCAAAA CTTACACTCT AACTGATTAC TTAAAAAAT 3', SEQ ID NO: 30) and primer DPP4-R 5' gtcggagcgg ccgcctaagg taaagagaaa cattgtttta tgaagtg 3' (SEQ ID NO: 31) with program 94°C for 45 sec initial denaturation, then 30 cycles of 94°C, 10 sec; 48^°C, 6 sec; 60°C, 4 min; followed by a 5 min extension at 72°C after cycling. The resultant PCR product is cleaved with restriction enzymes Sfil for 25 min at 50°C, then 1 hr with Notl at 37°C. The approx. 2.2 kb fragment is then inserted into pSecTag2-B vector cut with same, and transformed into bacteria under standard conditions. The resulting plasmid is #135.

Example 4.1: Mutation of FAP amino acid residue alanine 657 to aspartic acid: Aspartate 663 in DPPIV is one of a number of residues identified that is close to the valine and peptide bond ofthe bound inhibitor in the published crystal structure of DPPIV. It differs from the corresponding residue in wild type FAP alpha dimer enzyme (Ala 657 in FAP) suggesting that it is not critical for catalytic activity in this class of enzyme. This putative active site residue in FAP is replaced with the corresponding aspartate residue from DPPIV (D663). Replacement ofthe identified residue is done using standard overlap extension PCR. PCR primers for A657D mutation are forward mutagenic internal 5' tccagctggg aatattacGA Ctctgtctac acagagagat 13' (SEQ ID NO: 32); Reverse mutagenic internal: 5' AAT CTC TCT GTG TAG ACA GAG TCG TAA TAT TCC CAG CTG GA 3 ' (SEQ ID NO: 33); and two non-mutagenic flanking primers hFAP-RV-F (forward): 5' TAG ATG GAA ATT ACT TAT GGT ACA AGA TGA TTC TTC C 3' (SEQ ID NO: 34) (located ca.100- 120 nt upstream of unique EcoRV site near nt 1747-1782 of in hFAP sequence, Accession No. NM_004460); and hFAP-Not-R (reverse): 5' ggt cgc tea gcg gcc get tagtc tga caa aga gaa aca ctg ctt tag 3' (SEQ ID NO: 35) (with Notl restriction site (underlined) placed immediately after the stop codon). PCR mutagenesis is accomplished in two rounds: Round 1 : Tube A: 25 μl IX KOD

XL buffer, 0.2 μl KOD XL enzyme (Novagen, Madison, WI), 0.5 μl 10 uM hFAP-RV-F primer, 0.5 μl 10 uM A657D reverse primer. The template is 0.5 μl 1:500 dilution of plasmid #122 containing recombinant soluble hFAP in pSecTag2-B vector (InVitrogen) (approx. 0.5 ng). Tube B: 25 μl IX KOD XL buffer, 0.2 μl KOD XL enzyme (Novagen), 0.5 μl 10 μM hFAP-Not-R primer, 0.5 μl 10 μM A657D forward primer. The template is 0.5 μl 1 :500 dilution of plasmid #122 recombinant soluble hFAP in pSecTag2-B vector (InVitrogen) (approx. 0.5 ng). Round 2: 50 μl IX KOD buffer, 0.4 μl KOD XL enzyme (Novagen), 1 μl 10 μM hFAP-RV-F primer, 1 μl 10 μM hFAP-Not-R primer. The template is 0.5 μl each Round 1, Tube A and Tube B PCR reactions. Cycling parameters for both rounds are: initial denaturation at 94°C for 40 sec; then 25 cycles of 94°C for 15 sec, 54^°C for 15 sec and 72^°C for 1 min. After cycling, extension is continued at 72°C for 5 min followed by cooling to 4^°C. PCR products are isolated using a commercial kit (Qiagen), cut with EcoRV and Notl restriction enzymes, run on an agarose gel, and the approx. 600 nt fragment isolated using a commercially available kit (Qiagen). The recovered fragment is ligated to similarly-cut FAP- pSecTag2-B plasmid, to replace the wild-type fragment with the corresponding mutated fragment. Ligated DNA (0.5 microlitre) is transformed into commercially available E. coli electrocompetent cells and plated with ampicillin selection on LB plates. DNA minipreparations on resulting transformants are subjected to DNA sequencing using a commercial service to confirm the intended mutation. Techniques for these standard molecular biology procedures are familiar to those skilled in the art, and alternative strategies and techniques exist for accomplishing the same end. The resulting plasmid is named #233. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected. Production and testing of the recombinant enzyme is by methods described herein. The effect ofthe A657D mutation on selected properties of soluble FAP alpha dimer enzyme are shown in FIG. 12. FIG. 12A shows that the pH-activity profile is less sensitive to pH and is similar to DPPIV as shown in FIG. 13A, compared to the control plasmid #122. The latter (plasmid #122) shows the typical FAP alpha dimer enzyme pH profile reported in the literature. FIG. 12B shows that FAP mutant #233 is inhibited less strongly than the wild- type control #122. FIG. 12C shows that the degree of inhibition by val-boroPro is altered in the A657D mutant to be dependent on when the inhibitor is added relative to the substrate. However, plasmid #122 FAP is relatively insensitive to pre-inhibition. Pre-incubation of enzyme #233 with PT100 (FIG. 12C) renders it more inhibited than when substrate and inhibitor are added simultaneously. This indicates that slow-binding is occurring in mutant #233 and furthermore, that the presence of substrate protects from inhibitor binding, since the rate with simultaneous addition does not fall over time to the inhibited level seen with pre- incubation, even after 10 min into the assay. FIG. 12D (plasmid #122 control) and FIG. 12E (A657D mutant, plasmid #233) show activity versus Ala-Pro-AFC substrate concentration for Km determination. The scales are different because the Km is significantly lower in the A657D mutant (16 microMolar) compared to 490 microMolar in the control FAP alpha dimer enzyme from plasmid #122. Published values for full length WT FAP range from 200-460 microMolar (Sun et al., 2002, Protein Expr. Purif. 24, 274-281). The Km ofthe A657D mutant is very similar to that of DPPIV (FIG. 13D).

Unexpectedly, several significant properties are altered by the single A657D mutation, including decreased Km for synthetic substrate, broader pH optimum, an altered IC50 as well as slow-binding kinetics of proline boronic acid inhibitor val-boroPro, acquisition of sensitivity to inhibitor L-valine-2-nitrilo pyrollidine, and apparent irreversibility of inhibition by Val-boroPro. These coincide with desired improvements in FAP properties.

Example 4.2: Mutation of hFAP residues Y124H and A207S: The intended mutations are produced using overlap extension PCR. For Round 1 PCR

Tube A, the 5' PCR primer (Y124H-F: 5' TTTGTATATC TAGAAAGTGA TTATTCAAAG CTTTGGAGAC ACTCTTACACA G 3', SEQ ID NO: 36) overlaps the Xbal site and also serves to change the nearby Y124 to histidine, and the 3' reverse primer (A207S-R: 5' CCA GAG AGC ATA TTT TGT AGA AAG CAT TTC CTC TTC (SEQ ID NO: 37) overlaps and mutates A207 to serine. Tube B PCR has the mutagenic A207S primer in the forward sense (A207S-F: 5'gaagaggaaatgcttTctacaaaatatgctctctgg 3', SEQ ID NO: 38) and a reverse primer 3' to the unique Clal site. The products of PCR with these primers, using standard conditions, are mixed (0.5 μl each in a 50 μl reaction) and Round 2 PCR done with just the outermost primers Y124H-F and hFAP-Cla-R. The resulting PCR product of approx. 480 nt is purified and cut with Xbal and Clal, run on agarose and the band excised and DNA gel purified. This is then ligated to sr hFAP plasmid from which the corresponding Xbal -Clal fragment had been excised.

The resulting plasmid is named #217. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected.

Example 4.3: Mutation of hFAP residues A347V, G349R, F351R and V352P: The intended mutations are produced using overlap extension PCR, using standard conditions. For Round 1 PCR Tube A, the 5' forward primer (hFAP-Cla-F 5' CGG ATA TTT ATT ATC GAT ACC ACT TAC CCT GC 3', SEQ ID NO: 39) is paired with the mutagenic R356-R. primer (5' TGA AGG CCT AAA TCT TCC AAC CCA Tec agt tct get ttc ttc tat atgctcc 3', SEQ ID NO: 40). For Tube B the 5' PCR primer (R356-F: 5'

TGGGTTGGAA GATTTAGGCC Ttcaacacc agttttcag ctatgatg 3', SEQ ID NO: 41) is combined with the 3' reverse primer hFAP-RV-R (5' ctgtatttgctgttaat tgG ATA TCttaccttgcaagcacagaaaacatt 3', SEQ ID NO: 42). The products of Round 1 PCR with these primers are mixed (0.5 μl each in a 50 μl reaction) and Round 2 PCR done with just the outermost primers hFAP-Cla-F and hFAP-RV-R. The resulting PCR product of approximately 910 nt is purified and cut with Cla I and EcoRV, run on agarose and the approximately 875 nt band excised and DNA gel purified. This is then ligated to sr hFAP plasmid from which the corresponding Cla I-EcoRV fragment has been excised. The resulting plasmid is named #219. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected.

Example 4.4: Recombinant hFAP with mutations Y124H, A207S, A347V, G349R, F351R and V352P: Plasmids #217 and #219 from above examples are spliced together to combine the sets of mutations therein. The mutated Xba I - Cla I fragment from plasmid #217 is excised with the cognate enzymes and ligated to plasmid #219 from which the corresponding fragment has been excised. This gives a plasmid named #257. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected.

Example 4.5: Recombinant hFAP with mutations Q732E and N733D in a mouse/human FAP chimera: This alters two residues N-terminal to the catalytic histidine. The intended mutations are produced using overlap extension PCR using plasmid #23 containing chimeric FAP with N-terminal 77 amino acids from mouse FAP and the remainder human FAP with T229M mutation as template. For Round 1 PCR Tube A, the 5' PCR primer (hFAP-RV-F 5' TAGATGGAAA TTACTTATGG TACAAGATGA TTCTTCC 3 ', SEQ ID NO: 43) pairs with the mutagenic 3' reverse primer (DEDH-R 5' aatgtggtac tctgacGaAG accacggctt atccggcctg 1 3', SEQ ID NO: 44). Tube B PCR has the mutagenic (DEDH-F 5' tggtcttcgt cagagtacca cattgcctgg 3', SEQ ID NO: 45) primer in the forward sense and the reverse primer pSecTag-R (5' GGCGCTATTC AGATCCTCTT CTGAGAT 3', SEQ ID NO: 46). The products of PCR with these primers are mixed (0.5 μl each in a 50 μl reaction) and Round 2 PCR done with just the outermost primers. The resulting PCR product of approx. 760 nt is purified and cut with EcoRI and Not I, run on agarose and the approx. 250 nt gel band excised and DNA purified. This is then ligated to plasmid #23 (rs hFAP chimera with 77 N-terminal amino acids from mFAP) from which the corresponding EcoRI-Not I fragment has been excised. The resultant plasmid is designated #94 which is confirmed by DNA sequencing. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected. The IC50 of this mutant for Val-boroPro inhibitor with 0.1 mM Ala-Pro- AFC substrate is measured at 12 nM compared to 20 nM for parent FAP plasmid and <0.5 nM for recombinant soluble DPPIV produced from plasmid #135. FIG. 14 shows IC50 determination on soluble secreted hFAP alpha dimer enzyme with mutations Q732E and N733D (Plasmid #94) compared to "wild-type" FAP alpha dimer enzyme (#122) for val- boroPro and val-nitriloPro inhibitors. Both proteins include a T229M mutation relative to published hFAP. Curve land 2 are #94 and #122 with val-boroPro respectively; curve 3 and 4 are #94 and #122 with val-nitriloPro respectively. The results show that this double mutation lowers the IC50 of both inhibitors Val-boroPro and Val-nitriloPro. Example 4.6: Recombinant hFAP with mutations A347V, G349R, F351R and V352P, Q732E and N733D: Plasmids #94 and #219 from above examples are spliced together to combine the sets of mutations therein. The 3' end ofthe gene is excised from plasmid #94 with EcoRV and Notl and ligated to #219 cut with the same enzymes to give a plasmid #254. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected.

Example 4.7: Recombinant hFAP with mutations A347V, G349R, F351R and V352P and A657D: Mutated segments from plasmid #219 and #233 are combined using the restriction enzymes EcoRV and Notl to excise the A657D mutation from #233 for insertion into #219. The resultant plasmid with the combined mutations is called #245.

Example 4.8: Recombinant chimeric hFAP-DPPIV with N679-N733 replaced by the corresponding human DPPIV residues N685-D739: A unique EcoRI site overlaps the codons for amino acids 678-680 of human FAP. The region between this site and the histidine ofthe catalytic triad is replaced by the corresponding region of hDPPIV based on alignments ofthe sequences. This is accomplished in 2 stages. First the 3' end of hFAP from the EcoRI site to the 3' end is replaced by the corresponding region of DPPIV, then the C-terminal 24 amino acids of FAP are restored by overlap extension PCR using KOD thermostable polymerase (Novagen, Madison, WI, USA). The first step is accomplished by inserting a EcoRI - Notl PCR fragment made from a forward DPPIV primer (FAP-DPP4-RI-F: 5' ggatgataat cttgagcac tataaGAATT Caacagtcat gagcagagct 3', SEQ ID NO: 47) that inserts a EcoRI site at amino acid 684-686 of DPPIV and a reverse primer that places a Not I site immediately 3' ofthe DPPIV stop codon. After digestion with EcoRI and Notl, an approximately 250 nt fragment is gel purified and used to replace the EcoRI-Not I 3' fragment of sr hFAP/mouse FAP chimera in plasmid #23. This new plasmid #102, has no measurable enzymatic activity in supernatants of transfected mammalian cell line 293 T. Restoration of activity is accomplished by restoring the 3 ' 27 codons from hFAP C-terminus and the human N-terminal 77 amino acids, leaving an internal portion of 55 amino acids from DPPIV. The intended restoration is produced using overlap extension PCR. For Round 1 PCR Tube A, the template is plasmid #102 with the 5' PCR primer (FAP-DPP4-RI-F: 5' ggatgataat cttgagcac tataaGAATT Caacagtcat gagcagagct 3', SEQ ID NO: 47) paired with the mutagenic 3' reverse primer (DPP-FAP-R: 5' agg ccg gat aag ccA TGG TCT TCA TCA GTA TAC CAC ATT GCC TGG A 3', SEQ ID NO: 48). The latter has FAP homology at its 5' end and DPPIV homology at its 3' end, joining DPPIV D739 in-phase with hFAP H734. Tube B PCR, with hFAP as template, has the mutagenic (DPP-FAP-F 5' CAATGTGGTA TACTGATGAA GACCATggct tatccggcct gtccac 3', SEQ ID NO: 49) primer in the forward sense. This primer overlaps DPP-FAP-R. The reverse primer is hFAP-Not-R (5'ggt cgc tea gcg gcc get tagtc tga caa aga gaa aca ctg ctt tag 3', SEQ ID NO: 35). The products of Round 1 PCR are mixed (0.5 μl each in a 50 μl reaction) and Round 2 PCR done with just the outermost primers FAP-DPP4-RI-F and hFAP-Not-R. The resulting PCR product of ca 260 nt is purified and cut with EcoRI and Not I, run on agarose and the band excised and DNA gel purified. This is then ligated to sr hFAP plasmid #122 from which the corresponding fragment had been excised. The resultant plasmid is #155. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected which yields active enzyme.

Example 4.9: Recombinant chimeric hFAP-DPPIV with mutations A347V, G349R, F351R and V352P in the FAP portion and N679-N733 replaced by the corresponding human DPPIV residues: The mutation in plasmids #155 and #219 are combined by inserting the EcoR V-Not I restriction fragment from #155 into the corresponding location in plasmid #219 to give plasmid #251. DNA sequencing, large scale plasmid preparation and transfection of mammalian cells are done using standard procedures, and the cell culture supernatant containing the secreted enzyme is collected. FIG. 15 shows Eadie-Hoftsee plots for determination of Km for FAP-DPPIV chimera produced from plasmid #155 in tissue culture supernatant compared to control FAP (#122). The gradient is the negative value ofthe Km and is 417 and 561 microMolar for plasmid #155 and #122 respectively. Plasmid #122 serves as wild-type control, and the chimera has a lower Rm. Example 4.10: Recombinant soluble hDPPIV with D663A mutation: The intended mutations are produced using overlap extension PCR. The two overlapping mutagenic primers for this mutation are DPP4-A663-F (5' TCC CGG TGG GAG TAC TAT GCC TCA GTG TAC ACA GA 3', SEQ ID NO: 50), and DPP4-A663-R (5' TCT GTG TAC ACT GAG GCA TAG TAC TCC CAC CGG GA, SEQ ID NO: 51). The flanking non-mutagenic primers are DPPIV 1300-F (5' AAGACTGCAC ATTTATTACA AAAGGCACC 3', SEQ ID NO: 52) and DPP4-R (5' gtcggagcgg ccgcctaagg taaagagaaa cattgtttta tgaagtg 3', SEQ ID NO: 31). In Round 1, the template is plasmid #135 which contains DPPIV deleted for amino acids 1-38, fused to the immunoglobulin kappa chain secretory sequence in pSecTag2-B via an engineered Sfi I restriction site that leaves 6 vector- encoded amino acids at the N-terminus ofthe soluble protein (see Example above). In Round 2 the flanking primers generate an approx. 1.15 kb piece from the two Round 1 products. The PCR fragment and plasmid #135 are cut with BstX I and the internal approximately 625 nt fragment is replaced with the mutated fragment by ligating approximately 40 ng vector with 10 ng insert in a 5 microlitre ligation. The resultant plasmid after transformation and screening is #266. This plasmid is transfected into 293T fibroblast cells and secreted enzyme collected in the culture supernatant. Mutation of this residue in native, membrane-bound mouse DPPIV to alanine or glycine has been reported, however the mutation was characterized as not significantly modifying the expression or enzymatic properties ofthe resultant enzyme. (David, F. et al. 1993. J. Biol. Chem., 268, 17247-17252.). Therefore, the corresponding human DPPIV D663 (mouse D657) aspartate residue would be expected to be an unlikely candidate for altering enzymatic properties. However, the secreted form of human DPPIV D633A mutant described in this Example has novel properties that are significantly different from its parent. The effect ofthe D663 mutation on selected properties of soluble DPPIV are shown in FIG. 13. FIG. 13 A shows that the pH-activity profile is less sensitive to pH, compared to the control plasmid #135. The latter (plasmid #135) shows the typical DPPIV pH profile reported in the literature. FIG. 13B shows measurement of IC50 for Val-boroPro inhibitor in assays in which substrate and inhibitor were added simultaneously. A significant change in IC50 in #266 (arrow 1) compared to wild-type soluble DPPIV (arrow 2) is seen. Arrow 3 shows wild- type FAP for comparison. The DPPIV IC50 is abnormally high because pre-incubation with inhibitor was deliberately omitted, so that alterations in slow-binding kinetics would be apparent. FIG. 13C shows that the response to inhibition by val-boroPro is altered in the D663 A mutant to be more sensitive when inhibitor and substrate are added simultaneously as demonstrated by the greater difference in the slope ofthe curves for the wild-type depending on how long a pre-incubation was allowed before substrate addition, i.e. the slow-binding kinetics is diminished at least when inhibitor is added 30 sec before substrate, thereby becoming more similar to wild-type FAP. FIG. 13D shows activity versus Ala-Pro-AFC substrate concentration for Km determination for plasmid #135 (control) and D663A mutant, plasmid #266. Km values of 51 microMolar for mutant #266 and 14 microMolar for wild-type DPPIV (the latter indistinguishable from published values) were found. Thus, the Km for Ala- Pro-AFC substrate is altered. Second, the slow-binding property for proline-2-boronic acid inhibitors, typified by its response to Val-boroPro, is diminished if not eliminated. Furthermore, its pH profile shows a marked resemblance to that of FAP rather than DPPIV.

Example 4.11: Measurement of inhibition of mutant FAP dimer enzyme from plasmids #217, 219, 251, 255, 257, 233 and 245 by Val-nitriloPro compared to control FAP (#122) and DPPIV (#135): Soluble recombinant enzyme was produced in the supernatant medium from transfected HEK 293T cells. Assays were conducted as in Example 1.7 and contained Q.2mM Ala-Pro-AFC substrate. Production of fluorescence was monitored continuously for 20 min, and linear rates were extracted from the data. FIG. 11 shows the effect of FAP mutations described in the examples above on the percent inhibition by the inhibitor val-nitriloPro (valine-2-nitrilo-pyrollidine) at a range of concentrations. Soluble FAP from plasmid #217 shows little effect on inhibition compared to #122 which is used as a control. In contrast, all mutants containing the hFAP A347V, G349R, F351R and V352P mutations (plasmids 219, 251, 255, 257) show increased sensitivity to this inhibitor, in the range of 60-70% inhibition at 4 microMolar. The only exception is plasmid #245 which carries the additional A657D mutation. The latter (plasmid #245) and the A657D mutant #233, produce FAP alpha dimer enzymes with an even greater susceptibility more similar to DPPIV (e.g., approximately 90% at 4 microMolar). The responses of these FAP alpha dimer mutants at 4 microM Val-nitriloPro are summarized in Table 2, and can be roughly divided into 3 classes based on degree of inhibition. Less than 50% inhibition: wild type FAP and FAP mutant #217 (Y124H, A207S). 60-70% inhibition: FAP mutants containing the combined A347V, G349R, F351R, V352P set of mutations found in plasmid #219 and derivatives thereof #251, #254, and #257. Greater than 90% inhibition: wild type DPPIV (#135), and FAP mutants #233, #245 which both contain the A657D mutation.

Table 2. % inhibition of FAP mutants by val-nitriloPro at 4 micromolar Plasmid #217 #219 #233 #₂₄5 #₂51 #₂55 #₂57 #12₂ hFAP _h #J⁵ _|v % inhibition 45 65 95 95 65 70 68 43 100

Since plasmids #245 and #233 both share the A657D mutation, it thus inferred that this single amino acid change can significantly alter this particular property of FAP to resemble DPPIV. Mutation of this FAP residue to aspartate is a preferred embodiment, as is its mutation to any other amino acid but in particular to amino acids with less bulky side chains including glycine, serine, cysteine, and valine.

Example 4.12: Recombinant soluble hDPPIV with V354A, R356G, R358F, P359V mutations: The "R356" region of DPPIV constitutes a loop, the apex of which, based on crystal structure analysis, comprises the R357, F357 and R358 residues which are exposed in the interior ofthe active site. Four residues in this region are changed to the corresponding FAP residues using overlap extension PCR to make the loop more hydrophobic overall. The intended mutations are produced using overlap extension PCR. For Round 1 PCR, Tube A, the PCR primers are S wal-F ( 5 ' GAC ATTTATGATTTAAATAAAAGGC AGCTGATTAC AGAAGAG 3', SEQ ID NO: 53) and R356-R: 5' CTG AAG CGA AAA AAC CTC CAG CCC AGC CAG TAG TAC TCA TTC AAT G 3' (SEQ ID NO: 54) and Tube B contains primers R356-F: 5' GCTGGAGGTT TTTTCGCTTC AGAACCTCAT TTTACCCTTG ATGGT 3' (SEQ ID NO: 55) and DPPIV_BspEI-R sequencing primer (5' TAG TAC TGA CAC CTT TCC GGA TTC AGC TCA 3 ', SEQ ID NO: 56) with cloned DPPIV cDNA plasmid #135 as template. The products ofthe first reactions are combined with the external primers Swal-F and DPPIV_BspEI-R in Round 2, and the resulting product exit with Swal and BspEI giving an approx 940 nt fragment and ligated into sr hDPPIN plasmid #135 prepared with same enzymes. Cycling parameters for both rounds are: initial denaturation at 94°C for 40 sec; then 25 cycles of 94°C for 15 sec, 54°C for 15 sec and 72^°C for 1 min. After cycling, extension is continued at 72°C for 5 min followed by cooling to 4^°C. PCR products are isolated using a commercial kit (QIAquick PCR purification Kit, Qiagen), cut with Swa I and BspE I restriction enzymes, run on an agarose gel, and the approximately 600 nt fragment isolated using a commercially available kit (QIAquick Gel extraction Kit, Qiagen). Expression of soluble enzyme follows the protocol in the preceding examples for soluble recombinant proteins.

Example 4.13: Recombinant soluble hDPPIV with H126Y mutation: The H126Y mutation can be introduced in a single round of PCR due to proximity to a unique Swa I restriction site. A single reverse primer (H124Y-R: 5' GCCTTTTATTTAAAT CAT AAA TGT CAT ATG AAG CTG TGT AGG AAT aCC TCC ATT 3', SEQ ID NO: 57; Swa I site underlined), is coupled with the Sfi-DPP4 primer (5 ' GTAGTCGGCC CAGCCGGCC AGTCGCAAAA CTTACACTCT AACTGATTAC TTAAAAAAT 3', SEQ ID NO: 30) to generate a PCR fragment from DPPIV template (e.g. plasmid #135) that is digested with Sfi I-Swa I restriction enzymes and used to replace the corresponding fragment at the 5' end ofthe hDPPIV gene in plasmid #135 using standard ligation techniques.

Example 4.14: Recombinant soluble hDPPIV with S209A mutations: The intended mutation is produced using overlap extension PCR. For Round 1 PCR, Tube A, the PCR primers are Swal-F (5' GACATTTATG ATTTAAATAA AAGGCAGCTG ATTACAGAAG AG 3', SEQ ID NO: 53) and S209A-R (5' ACC ACC ACA GAG CAG CGT AGG CAC TGA AGA CT 3', SEQ ID NO: 58) and Tube B contains primers S209A-F (5' AGTCTTCAGT GCCTACTaTG CTGTGTGGTG GT 3', SEQ ID NO: 59) and DPPIV_BspEI-R sequencing primer (5' TAG TAC TGA CAC CTT TCC GGA TTC AGC TCA 3', SEQ ID NO: 56) with cloned DPPIV cDNA in plasmid #135 as template. The products ofthe first reactions are combined with the external primers Swal-F and

DPPIV_BspEI-R in Round 2, and the resulting product cut with Swal and BspEI giving an approx 940 nt fragment and ligated into sr hDPPIN plasmid #135 prepared with the same enzymes.

Example 5.1: Soluble recombinant human-mouse FAP chimera with Ν-terminal 77 amino acids (excludes vector-derived residues) from mouse FAP: The Sfi I-Xbal fragment of plasmid #13 containing the entire coding region of human FAP, but with a single T229M amino acid change, is replaced by the corresponding fragment from mouse FAP, generated by PCR. The primer Sfi-FAP-B (5' GTAGTCGGCC CAGCCGGCCA CAAAGAGAGC TCTTACCCTG AAGGATATTT TAAATG 3', SEQ ID NO: 13) and a reverse primer mFAP45 (5' TTC CAT TGG GCC CAC GTG GTG 3', SEQ ID NO: 60) located 3' ofthe conserved Xba I site (the latter overlaps amino acids 113-115) , were used to amplify the 5' end ofthe mouse gene between amino acids 38 -115. The PCR product is digested with Sfi I and Xba I restriction enzymes and inserted into plasmid #13 from which the 5' end ofthe gene is excised with the same enzymes. The resultant plasmid fuses the vector-encoded immunoglobulin secretion sequence to codon #39 ofthe mouse FAP which is, in turn fused to the human FAP at amino acid 114. The resulting plasmid, called #23, contains 77 amino acids of mouse FAP and 683 amino acids of human FAP. Because of homology between the two species, there are 13 amino acid differences in the chimeric segment compared to the wholly human FAP alpha dimer enzyme.

Example 5.2: Soluble recombinant human-mouse FAP chimera with N-terminal 77 amino acids only (excludes vector-derived residues) from human FAP: A plasmid pcDNA FAP#5 containing mouse FAP cDNA corresponding to the published sequence (GenBank Accession number Y10007) is obtained from a commercial source cloned into the poly linker of a commercially available vector pcDNA (InVitrogen). This cDNA is excised using EcoRI and Notl restriction sites in the flanking polylinker and ligated to pSecTag2-B secretion vector (InVitrogen), giving plasmid #18. A truncated approximately 260 nt 5' fragment of human FAP deleting the first 38 amino acids of hFAP and inserting a Sfi I restriction site adjacent to the Thr39 codon is derived by PCR. This allows an in-phase junction to the vector Ig-kappa secretion sequence at the Sfi I site. The 5' PCR primer (Sfi-FAP-B 5' GTAGTCGGCC CAGCCGGCCA CAAAGAGAGC TCTTACCCTG AAGGATATTT TAAATG 3', SEQ ID NO: 13 (Sfi I site underlined) makes residue #39 a lysine, as found in mouse FAP. PCR of FAP cDNA with this primer and a reverse primer (FAP porbe 5' tgaaataataGtcacttgaggctatcatt 3') located 3' of the common, conserved unique Xba I site, gives a PCR product of approximately 700 nt. A Sfi I-Xba I double digest on plasmid #18 is used to remove the native 5' end of mouse FAP up to the internal Xba I site at codon 114. and to cut the FAP PCR product. The appropriate fragments of >5 kb and approximately 259 nt respectively are isolated from an agarose gel, ligated, and transformants screened. The resulting plasmid, called #29, contains 77 amino acids of human FAP and 683 amino acids of mouse FAP. Because of homology between the two species, there are a total of 13 amino acid differences in the chimeric segment compared to the wholly mouse FAP. The N-terminus ofthe final mature a. a. sequence of cleaved secreted product will contain 6 a.a from the vector, DAAQPA (SEQ ID NO: 14), fused to the truncated FAP sequence, of which the first 13 amino acids are TKRALTLKDILNG (SEQ ID NO: 15). FIG. 9 shows soluble secreted FAP alpha dimer activity in tissue culture supernatant from plasmids #23, #29 and #43 measured by production of fluorescence from Ala-Pro-AFC substrate at pH 8.1.

Example 5.3: Production of soluble secreted mouse FAP: The approximately 250 nt Sfil-Xbal 5' fragment of plasmid #23 is ligated to similarly-cut plasmid #29 to generate a plasmid #43 for production of soluble mouse FAP amino acids 39-760 of which the first 13 amino acids are TKRALTLKDILNG (SEQ ID NO: 15). The N-terminus ofthe cleaved mature secreted mouse FAP will contain 6 amino acid from the vector (i.e., DAAQPA, SEQ ID NO: 14). This plasmid produces soluble secreted recombinant FAP dimer activity when expressed in HEK 293T cells, as shown in FIGs. 5 and 9.

Example 5.4: Soluble human FAP chimera with amino acids #269-557 substituted with the corresponding mouse FAP residues: The Clal - EcoRV region of sr hFAP in plasmid #122 is replaced with a PCR fragment of mouse FAP containing the corrresponding region. The PCR primers are designed to introduce the Clall - EcoRV sites into the mouse fragment to facilitate cloning. The resulting plasmid, #279, gives low but measurable FAP activity. The residues in murine wild type FAP alpha dimer enzyme correspond to those in the human homolog.

Example 5.5: Soluble human FAP chimera with amino acids # 269-448 substituted with the corresponding mouse FAP residues: The region noted corresponds to the Cla I - Ban I region of sr hFAP in plasmid #122, which is replaced with a PCR fragment of wild type mouse FAP alpha dimer enzyme containing the corresponding amino acids. Since mFAP does not have a Clal restriction site, PCR primers were designed to a chimeric Clal-EcoRV fragment (FAP residues introduce the Cla I site into the mouse fragment to facilitate cloning. The resulting plasmid, #286, gives good FAP enzymatic activity. The residues in murine wild type FAP alpha dimer enzyme correspond to those in the human homolog.

Example 5.6: Soluble human FAP chimera with mouse FAP amino acids # 449-557: This hFAP-mouse chimera corresponds to replacement ofthe Banl - EcoRV fragment of hFAP (amino acids # 449-557) with the mouse equivalent is made by creating a chimeric Clal-EcoRV fragment by PCR. This is accomplished by digesting plasmid #122 (sr hFAP) with Clal and EcoRV, isolating the approximately 0.9 kb fragment, mixing it with plasmid #264 (sr hFAP with Clal-EcoRV from mouse) and jointly cutting with Banl. DNA is purified from the digest with a kit (QIAquick PCR purification, Qiagen) and self ligated. Chimeric molecules ligated at the Banl site are selected by PCR with a 5' human primer hFAP-Cla-F and a mouse reverse primer mFAP-RV-R. The PCR product is digested with Clal and EcoRV and ligated to similarly-cut plasmid #122. This gives plasmid #279. A map of this plasmid is shown in FIG. 16A and its activity is shown in FIGs. 16B and 16C relative to other mouse- human chimeras.

Example 5.7: Soluble human FAP chimera with mouse FAP amino acids # 449-557 with reversion of mouse region K512 - F518 to the human residues: The hFAP-mouse Banl - EcoRV chimera (plasmid #279) is altered to restore the human residues E512 - L518 using overlap-extension PCR with #279 as template. The overlapping forward and reverse mutagenic PCR primers were designed to introduce the desired changes into separate overlapping halves ofthe mouse fragment which is then joined in the second round of PCR, cut with Clal and EcoRV. The resulting plasmid, #326, gave improved FAP activity over #279, indicating that changes in these 7 residues in human FAP were not well tolerated, at least in the context of a human-mouse chimera. A map of representative human-mouse chimeras of soluble FAP is shown in FIG. 16A. These show the relevant mouse and human portions ofthe chimeras. In the case of #319 and #326, the location of additional point mutations is shown. All are derived from plasmid #122 and so also carry the T229M change. The relative activity of tissue culture supernatants of representative hu-mouse FAP chimeras Cla I-EcoRV interval are shown in FIG. 16B and 16C. The reversion of mouse region K512 - F518 to the human residues in #326 has a dramatic effect on resultant activity. In all cases, the same amount of plasmid was used to transfect HEK 293T cells and the an aliquot ofthe supernatants assayed with Ala-Pro-AFC.

Example 6.1: Soluble forms treated with Val-boroPro or other proline-2-boronic acid that slowly regain activity ("Slow release"): Soluble recombinant DPPIV or its D663A mutant in a pharmaceutically acceptable injectable preparation is treated with Val-boroPro at a concentration of approximately 0.5 nM or higher but typically not greater than 10 micromolar, for a period of approximately 1 -15 min or longer, during which time strong inhibitory binding ofthe inhibitor occurs to the wild- type enzyme or relative strong binding in the case ofthe D663A mutant.

Example 6.2: Inhibitor-bound recombinant soluble FAP A657D mutant with slow- release of activity: Soluble recombinant FAP A657D mutant in a pharmaceutically acceptable injectable preparation is treated with Val-boroPro at a concentration of approximately 0.5 nM or higher but typically not greater than 10 micromolar, for a period of approximately 1 -15 min or longer, during which time a complex of intermediate duration forms.

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32. De Meester, I., Korom, S., Van Damme, J. & Scharpe, S. CD26, let it cut or cut it down. Immunol. Today 20, 367-375 (1999).

33. Marguet, D. et al. Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26. Proc. Natl. Acad. Sci. USA 91, 6874-6879 (2000). 34. Mentlein, R., Gallwitz, B. & Schmidt, W.E. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide- 1 (7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Ewr. J. Biochem. 214, 829-835 (1993).

35. Park, J.Ε. et al. Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J. Biol. Chem. 274, 36505- 36512 (1999).

36. Garin-Chesa, P., Old, L.J. & Rettig, W.J. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cells. Proc. Natl. Acad. Sci. USA 87, 7235-7239 (1990). 37. von Bonin, A., Huhn, J. & Fleischer, B. Dipeptidyl-peptidase IV/CD26 on T cells: analysis of an alternative T-cell activation pathway. Immunological Reviews 161, 43- 53 (1998). 38. Yaron, A. & Νaider, F. Proline-dependent structural and biological properties of peptides and proteins. Crit. Rev. Biochem. Mol. Biol 28, 31-81 (1993). 39. Demuth, H.-U. & Heins, J. Catalytic mechanism of dipeptidyl peptidase IV. in Dipeptidyl peptidase IV (CD26) in metabolism and immune response (ed. Fleischer, B.) 1-34 (R.G. Landes Co., 1995). 40. Courts, S.J. et al. Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation ofthe P₂ position of X_aa-boroPro dipeptides. J. Med. Chem. 39, 2087-2094 (1996).

41. Tsuji, E. et al. An active site mutation (Gly to Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum. Biochemistry 31, 11921-11927 (1992).

42. Dinarello, CA. The biological properties of interleukin-1. Ewr. Cytokine Net. 5, 517- 531 (1994). Equivalents The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope ofthe invention. Various modifications ofthe invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope ofthe appended claims. The advantages and objects ofthe invention are not necessarily encompassed by each embodiment ofthe invention. All references, patents and patent applications that are recited in this application are incorporated by reference herein in their entirety.

What is claimed is:

Claims

1. A method for down-regulating an immune response comprising administering to a subject in need thereof a FAP alpha dimer enzyme in an amount effective to down-regulate an immune response.

2. The method ofclaim 1, wherein the immune response is an IL-1 mediated condition. 3. The method of claim 1 , wherein the immune response is an abnormal immune response.

4. The method of claim 3, wherein the abnormal immune response is selected from the group consisting of inflammation, autoimmune disease, sepsis, graft versus host disease, transplant rejection, toxic shock syndrome, allergy, asthma, atherosclerosis, osteoarthritis, and Guillain-Barre's syndrome.

5. The method of claim 3, wherein the abnormal immune response is subsequent to an infection.

6. The method of claim 5, wherein the infection is an RSV infection.

7. The method of claim 4, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin dependent diabetes (type I diabetes), inflammatory bowel disease, autoimmune thyroiditis, systemic lupus erythematosus (SLE), uveitis, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre's syndrome, psoriasis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis and multiple sclerosis. 8. The method of claim 1 , wherein the subject does not have cancer or the subject does not have a predisposition to cancer.

9. The method of claim 1 , further comprising administering to the subject a second agent.

10. The method of claim 9, wherein the second agent is an anti-inflammatory agent.

11. The method of claim 9, wherein the second agent is an immunosuppressant.

12. The method of claim 9, wherein the second agent is an anti-infective agent.

13. The method of claim 12, wherein the anti-infective agent is an anti-bacterial agent.

14. The method of claim 12, wherein the anti-infective agent is an anti- viral agent.

15. The method of claim 12, wherein the anti-infective agent is an anti-fungal agent.

16. The method ofclaim 12, wherein the anti-infective agent is an anti-parasitic agent.

17. The method of claim 12, wherein the anti-infective agent is an anti- mycobacterial agent. 18. The method of claim 1 , wherein the FAP alpha dimer enzyme is wild type FAP alpha dimer enzyme.

19. The method of claim 1 , wherein the FAP alpha dimer enzyme is a truncation mutant.

20. The method of claim 1 , wherein the FAP alpha dimer enzyme is a fusion or chimeric protein.

21. The method of claim 20, wherein the fusion or chimeric protein comprises a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a self-cleaving affinity tag, a tissue localization sequence and a peptide or protein ligand.

22. The method of claim 21 , wherein the secretion sequence is a G-CSF leader sequence or an Ig-kappa leader sequence. 23. The method of claim 21, wherein the purification sequence is selected from the group consisting of a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain.

24. The method of claim 21, wherein the epitope is selected from the group consisting of a hemaglutinnin tag, a FLAG tag, a V5 tag, a myc tag and a T7 tag.

25. The method ofclaim 21, wherein the protein degradation sequence is a PEST sequence. 26. The method of claim 21 , wherein the protease cleavage site is selected from the group consisting of enterokinase, factor Xa protease, thrombin, TEV protease, PreScission protease, Furin, and Genenase.

27. The method of claim 20, wherein the fusion or chimeric protein comprises an amino acid substitution of Q732E or N733D .

28. The method of claim 1 , wherein the FAP alpha dimer enzyme is a heterodimer.

29. The method of claim 28, wherein the heterodimer is a heterodimer of a FAP alpha monomer and a DPPIV/CD26 monomer.

30. The method ofclaim 1, wherein the FAP alpha dimer enzyme comprises an amino acid substitution relative to wild type FAP alpha dimer.

31. The method of claim 30, wherein the amino acid substitution is present in the β-propeller domain.

32. The method ofclaim 30, wherein the amino acid substitution is selected from the group consisting of Y124, A207, A347, G349, F351, V352. 33. The method of claim 30, wherein the amino acid substitution is selected from the group consisting of Y124H, A207S, A347V, G349R, F351R, V352P.

34. The method of claim 30, wherein the amino acid substitution is present in the catalytic domain.

35. The method of claim 34, wherein the amino acid substitution is in amino acid A657.

36. The method of claim 30, wherein the amino acid substitution is A657D.

37. The method of claim 30, wherein the amino acid substitution is Y124H or A207S.

38. The method of claim 30, wherein the amino acid substitution is A347V, G349R, F351R or V352P.

39. The method of claim 30, wherein the amino acid substitution is present in the entrance to the catalytic site. 40. The method of claim 39, wherein the entrance to the catalytic site is an apical entrance.

41. The method of claim 40, wherein the amino acid substitution is selected from the group consisting of G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K and Y458E. 42. The method of claim 39, wherein the entrance is a side entrance.

43. The method ofclaim 42, wherein the amino acid substitution is selected from the group consisting of N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, S119V, L121Q and Y124H.

44. The method ofclaim 30, wherein the amino acid substitution is present at an N-linked glycosylation site. 45. The method of claim 44, wherein the N-linked glycosylation site is selected from the group consisting of N49, N92, N99, N227, N314 and N679.

46. The method of claim 44, wherein the amino acid substitution is at T51 , T94, S101, T229, S316 or T681.

47. The method of claim 44, wherein the amino acid substitution is at N227 and T229.

48. The method of claim 44, wherein the amino acid substitution is T229M.

49. The method of claims 1-47 or 48, wherein the FAP alpha dimer enzyme is soluble.

50. The method ofclaim 30, wherein the amino acid substitution alters disulfide bond formation.

51. The method of claim 50, wherein the amino acid substitution introduces a disulfide bond.

52. The method of claim 51 , wherein the amino acid substitution is selected from the group consisting of H378C and A386C

53. The method of claim 51 , wherein the amino acid substitution is selected from the group consisting of L48C, N742C, M683C and I713C. 54. The method of claim 50, wherein the amino acid substitution removes a disulfide bond.

55. The method ofclaim 1, wherein the FAP alpha dimer enzyme is PEGylated. 56. The method of claim 55, wherein the FAP alpha dimer enzyme is PEGylated at a lysine.

57. The method of claim 55, wherein the FAP alpha dimer enzyme is PEGylated at a cysteine.

58. The method of claim 55, wherein the FAP alpha dimer enzyme is PEGylated at a cysteine introduced at position 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, 753, 91, 148, 263, 323, 343, or 444.

59. The method of claim 55, wherein the PEGylated FAP alpha dimer enzyme comprises a mutation in one or more amino acid positions selected from a group consisting of 95, 161, 173, 191, 219, 334, 372, 382, 436, 437, 445, 460, 486, 492, 499, 505, 509, 510, 521, 532, 533 564, 583, 591, 606, 616, 642, 670, 678, 715, 753, 91, 148, 263, 323, 343 and 444.

60. The method of claim 1, wherein the FAP alpha dimer enzyme is a dimerization domain mutant.

61. The method of claim 60, wherein the dimerization domain mutant lacks residues comprised of P232-I250 of wild type FAP alpha dimer enzyme and comprises residues P234-V254 of wild type DPPIV.

62. The method of claim 41 , wherein the dimerization domain mutant lacks residues F706-D731 of wild type FAP alpha dimer enzyme or some portion thereof and comprises residues F713-D738 of wild type DPPIV or some portion thereof. 63. The method of claim 60, wherein the dimerization domain mutant comprises an amino acid substitution of T248C

64. The method of claim 1, wherein the FAP alpha dimer enzyme lacks residues N679-N733 from wild type FAP alpha dimer enzyme and comprises residues N685-D739 of wild type DPPIV.

65. The method of claim 30, wherein the amino acid substitution is present in the cytoplasmic domain. 66. The method of claim 30, wherein the amino acid substitution is present in the transmembrane domain.

67. The method of claim 1 , wherein the FAP alpha dimer enzyme lacks a cytoplasmic domain.

68. The method of claim 1 , wherein the FAP alpha dimer enzyme lacks a transmembrane domain.

69. The method of claim 1 , wherein the FAP alpha dimer enzyme lacks a cytoplasmic and transmembrane domain.

70. The method of claim 1 , wherein the FAP alpha dimer enzyme lacks residues corresponding to 1-37 from wild type FAP alpha dimer enzyme.

71. The method of claim 1 , wherein the FAP alpha dimer enzyme is soluble.

72. The method ofclaim 71, wherein the FAP alpha dimer enzyme comprises an amino acid substitution of T229M.

73. The method ofclaim 1, wherein the FAP alpha dimer enzyme comprises an amino acid sequence of SEQ ID NO: 4.

74. The method of claim 1 , wherein the FAP alpha dimer enzyme is administered as a protein. 75. The method of claim 1, wherein the FAP alpha dimer enzyme is administered as a nucleic acid.

76. The method of claim 2, wherein the IL-1 is IL-1 alpha or IL-1 beta. 11. A composition comprising a FAP alpha dimer enzyme in a pharmaceutically acceptable carrier, wherein the composition is sterile and lacks an adjuvant.

78. A composition comprising a FAP alpha dimer enzyme in a pharmaceutically acceptable carrier, and a non-adjuvant second agent.

79. The composition ofclaim 78, wherein the non-adjuvant second agent is an anti-inflammatory agent.

80. The composition of claim 78, wherein the non-adjuvant second agent is an immunosuppressant.

81. The composition of claim 78, wherein the preparation is sterile.

82. The composition ofclaim 77 or 78, wherein the FAP alpha dimer enzyme is wild type FAP alpha dimer enzyme.

83. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme is a truncation mutant. 84. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme is a fusion or chimeric protein.

85. The composition of claim 84, wherein the fusion or chimeric protein comprises a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a tissue localization sequence, a peptide or protein ligand.

86. The composition of claim 85, wherein the secretion sequence is a G-CSF leader sequence or an Ig-kappa leader sequence.

87. The composition ofclaim 85, wherein the purification sequence is selected from the group consisting of a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain. 88. The composition of claim 85, wherein the epitope is selected from the group consisting of a hemaglutimiin tag, a FLAG tag, a V5 tag, a myc tag and a T7 tag.

89. The composition of claim 85, wherein the protein degradation sequence is a PEST sequence.

90. The composition of claim 85, wherein the protease cleavage site is selected from the group recognized by enterokinase, factor Xa protease, thrombin, TEV protease, PreScission protease, Furin, Genenase. 91. The composition of claim 84, wherein the fusion or chimeric protein comprises an amino acid substitution of Q732E orN733D.

92. The composition ofclaim 77 or 78, wherein the FAP alpha dimer enzyme is a heterodimer.

93. The composition of claim 92, wherein the heterodimer is a heterodimer of a FAP alpha monomer and a DPPIV/CD26 monomer.

94. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme comprises an amino acid substitution relative to wild type FAP alpha dimer.

95. The composition of claim 77 or 78, wherein the amino acid substitution is present in the β-propeller domain. 96. The composition of claim 95, wherein the amino acid substitution is at Y124,

A207, A347, G349, F351, V352.

97. The composition of claim 95, wherein the amino acid substitution is selected from the group consisting of Y124H, A207S, A347V, G349R, F351R, V352P.

98. The composition of claim 77 or 78, wherein the amino acid substitution is present in the catalytic domain.

99. The composition of claim 94, wherein the amino acid substitution is selected from the group consisting of Y124H, A207S, A347V, G349R, F35 IR, V352P and A657D.

100. The composition of claim 96, wherein the amino acid substitution is at A657.

101. The composition of claim 100, wherein the amino acid substitution is A657D.

102. The composition of claim 94, wherein the amino acid substitution is Y124H or A207S.

103. The composition of claim 94, wherein the amino acid substitution is A347V, G349R, F351R or V352P. 104. The composition of claim 94, wherein the amino acid substitution is present in the entrance to the catalytic domain.

105. The composition of claim 104, wherein the entrance to the catalytic domain is an apical entrance.

106. The composition ofclaim 105, wherein the amino acid substitution is selected from the group consisting of G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K and Y458E. 107. The composition of claim 104, wherein the entrance is a side entrance.

108. The composition ofclaim 104, wherein the amino acid substitution is selected from the group consisting of N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, S119V, L121Q and Y124H.

109. The composition of claim 94, wherein the amino acid substitution is present at an N-linked glycosylation site. 110. The composition of claim 94, wherein the N-linked glycosylation site is selected from the group consisting of N49, N92, N99, N227, N314 and N679.

111. The composition of claim 110, wherein the amino acid substitution is T229M.

112. The composition of claim 109, 110 or 111 , wherein the FAP alpha dimer enzyme is soluble.

113. The composition of claim 94, wherein the amino acid substitution alters disulfide bond formation.

114. The composition ofclaim 113, wherein the amino acid substitution introduces a disulfide bond.

115. The composition of claim 114, wherein the amino acid substitution is selected from the group consisting of H378C and A386C 116. The composition of claim 114, wherein the amino acid substitution is selected from the group consisting of L48C, N742C, M683C and I713C

117. The composition of claim 113, wherein the amino acid substitution removes a disulfide bond.

118. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme is PEGylated.

119. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme is a dimerization domain mutant.

120. The composition of claim 119, wherein the dimerization domain mutant lacks residues P232-I250 of wild type FAP alpha dimer enzyme and comprises residues P234-V254 of wild type DPPIV.

121. The composition of claim 119, wherein the dimerization domain mutant lacks residues F706-D731 of wild type FAP alpha dimer enzyme and comprises residues F713- D738 of wild type DPPIV. 122. The composition of claim 119, wherein the dimerization domain mutant comprises an amino acid substitution of T248C

123. The composition ofclaim 77, 78 or 84, wherein the FAP alpha dimer enzyme lacks residues N679-N733 from wild type FAP alpha dimer enzyme and comprises residues N685-D739 of wild type DPPIV.

124. The composition ofclaim 94, wherein the amino acid substitution is present in the cytoplasmic domain. 125. The composition of claim 94, wherein the amino acid substitution is present in the transmembrane domain.

126. The composition ofclaim 77, 78 or 83, wherein the FAP alpha dimer enzyme lacks a cytoplasmic domain.

127. The composition of claim 77, 78 or 83, wherein the FAP alpha dimer enzyme lacks a transmembrane domain.

128. The composition of claim 77, 78 or 83, wherein the FAP alpha dimer enzyme lacks a cytoplasmic and transmembrane domain.

129. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme lacks residues corresponding to 1-37 from wild type FAP alpha dimer enzyme. 130. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme is soluble.

131. The composition of claim 130, wherein the FAP alpha dimer enzyme comprises an amino acid substitution of T229M.

132. The composition of claim 77 or 78, wherein the FAP alpha dimer enzyme comprises an amino acid sequence of SEQ ID NO: 4.

133. The composition of claim 77 or 78, wherein FAP alpha dimer enzyme is present in an amount effective to down-regulate an immune response. 134. The composition comprising a FAP alpha dimer enzyme comprising an amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 70, and optionally (1) one or more amino acid substitutions selected from the group consisting of Y124H, A207S, A347V, G349R, F351R, V352P, A657D, Q732E, N733D, G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K, Y458E, N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, S116Y, D117N, SI 19V, L121Q, Y124H, H378C, A386C, L48C, N742C, M683C, I713C and T248C, (2) lacking residues P232-I250 and comprising residues P234-V254 of wild type DPPIV, (3) lacking residues F706-D731 and comprising residues F713-D738 of wild type DPPIV, (4) lacking residues N679-N733 and comprising residues N685-D739 of wild type DPPIV, or (5) an amino acid substitution of T229M.

135. The composition ofclaim 134, wherein the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 N-terminal amino acids in SEQ ID NO: 61 are deleted. 136. The composition of claim 135, wherein the FAP alpha dimer enzyme is a fusion or chimeric protein.

137. The composition of claim 136, wherein the fusion or chimeric protein comprises a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a self-cleaving affinity tag, a tissue localization sequence and a peptide or protein ligand.

138. The composition of claim 137, wherein the secretion sequence is a G-CSF leader sequence or an Ig-kappa leader sequence. 139. The composition of claim 137, wherein the purification sequence is selected from the group consisting of a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain.

140. The composition ofclaim 137, wherein the epitope is selected from the group consisting of a hemaglutimiin tag, a FLAG tag, a V5 tag, a myc tag and a T7 tag.

141. The composition of claim 137, wherein the protein degradation sequence is a PEST sequence. 142. The composition ofclaim 137, wherein the protease cleavage site is selected from the group consisting of enterokinase, factor Xa protease, thrombin, TEV protease, PreScission protease, Furin, and Genenase.

143. The composition of claim 134 or 135, wherein the FAP alpha dimer enzyme is a heterodimer.

144. The composition ofclaim 143, wherein the heterodimer is a heterodimer of a FAP alpha monomer and a DPPIV/CD26 monomer. 145. The composition of claim 134 or 135, wherein the amino acid substitution is

A657D.

146. The composition ofclaim 134 or 135, wherein the amino acid substitution is Y124H or A207S.

147. The composition of claim 134 or 135, wherein the amino acid substitution is A347V, G349R, F351R or V352P.

148. The composition of claim 134 or 135, wherein the amino acid substitution is selected from the group consisting of G64D, Q65H, V299A, D301Q, T354E, V356H, S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K and Y458E. > 149. The composition ofclaim 134 or 135, wherein the amino acid substitution is selected from the group consisting of N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, SI 16Y, D117N, SI 19V, L121Q and Y124H.

150. The composition ofclaim 134 or 135, wherein the FAP alpha dimer enzyme is soluble.

151. The composition of claim 134 or 135, wherein the FAP alpha dimer enzyme lacks residues P232-I250 and comprises residues P234-V254 of wild type DPPIV.

152. The composition of claim 134 or 135, wherein the dimerization domain mutant lacks residues F706-D731 and comprises residues F713-D738 of wild type DPPIV. 153. The composition of claim 134 or 135, wherein the FAP alpha dimer enzyme lacks residues N679-N733 and comprises residues N685-D739 of wild type DPPIV.

154. A composition comprising a FAP alpha dimer enzyme comprising an amino acid substitution of A657D.

155. The composition of claim 154, wherein the FAP alpha dimer enzyme is soluble.

156. The composition ofclaim 154, wherein the FAP alpha dimer enzyme further comprises an amino acid substitution of T229M. 157. The composition of claim 154, wherein the FAP alpha dimer enzyme further comprises an amino acid substitution of Y124H or A207S.

158. The composition of claim 154, wherein the FAP alpha dimer enzyme further comprises an amino acid substitution of A347V, G349R, F351R or V352P.

159. The composition ofclaim 154, wherein the FAP alpha dimer enzyme is a fusion or chimeric protein.

160. The composition of claim 159, wherein the fusion or chimeric protein comprises a sequence selected from the group consisting of a secretion sequence, a purification sequence, an epitope, a linker, a protein degradation sequence, a protease cleavage site, a self-cleaving affinity tag, a tissue localization sequence and a peptide or protein ligand. 161. The composition of claim 160, wherein the secretion sequence is a G-CSF leader sequence or an Ig-kappa leader sequence.

162. The composition of claim 160, wherein the purification sequence is selected from the group consisting of a GST sequence tag, a hexahistidine or polyhistidine tag, a Protein A tag, a biotin tag, a chitin tag, and a maltose binding domain.

163. The composition ofclaim 160, wherein the epitope is selected from the group consisting of a hemaglutinnin tag, a FLAG tag, a V5 tag, a myc tag and a T7 tag. 164. The composition of claim 160, wherein the protein degradation sequence is a

PEST sequence.

165. The composition ofclaim 160, wherein the protease cleavage site is selected from the group consisting of enterokinase, factor Xa protease, thrombin, TEV protease, PreScission protease, Furin, and Genenase. 166. The composition of claim 160, wherein the FAP alpha dimer enzyme is a heterodimer.

167. The composition of claim 166, wherein the heterodimer is a heterodimer of a FAP alpha monomer and a DPPIV/CD26 monomer.

168. The composition of claim 160, wherein the amino acid substitution is A347V, G349R, F351R or V352P.

169. The composition ofclaim 160, wherein the FAP alpha dimer enzyme further comprises an amino acid substitution of G64D, Q65H, V299A, D301Q, T354E, V356H,

S358T, Y359L, F401E, R402A, V403L, Q405S, T452S, A453V, D457K or Y458E.

170. The composition of claim 160, wherein the FAP alpha dimer enzyme further comprises an amino acid substitution of N49K, G50N, F52Y, S53R, Y54L, T56L, F57Y, F58S, P59L, S71Q, D73E, S91E, R93S, M95F, K96D, S97E, V98F, N99G, A100H, SI 16Y, D117N, SI 19V, L121Q or Y124H.

171. The composition of claim 160, wherein the FAP alpha dimer enzyme lacks residues P232-I250 and comprises residues P234-V254 of wild type DPPIV.

172. The composition of claim 160, wherein the dimerization domain mutant lacks residues F706-D731 and comprises residues F713-D738 of wild type DPPIV.

173. The composition of claim 160, wherein the FAP alpha dimer enzyme lacks residues N679-N733 and comprises residues N685-D739 of wild type DPPIV.

174. A composition comprising a FAP alpha dimer enzyme lacking amino acids 269-448 and comprising amino acids from mouse FAP.