Methods for Optimizing CATS Immunotherapeutics into Humanized Derivatives with Reduced Autoreactivity, Reduced Toxicity and Enhanced Long-Term Efficacy

Abstract

The present invention describes unique means for reducing autoreactivity that sensitizes against sustained treatments, and toxicity associated with administration of biologicals, in order to develop safer cancer immunotherapeutics. Short functional sequences are identified in the biologic and matched to their most homologous human counterpart. The human homologs are swapped in to replace their foreign counterparts. Alternatively, variants are selected that have exhibit less toxicity in human or primate experiments from nature, and these sequences are swapped in to replace their counterparts in the biologic. Also, human sequences that mediate the same function as foreign sequences in the biologic, but lack any sequence homology, can be swapped in for their foreign sequence counterparts. These inventions are applied to derivatize the HIV/SIV Tat protein into humanized trimers (CATS) useful for the treatment of cancer.

Claims

1- A humanized immunotherapeutic (CATS) consisting of primary sequence tiles discovered and characterized in this filing, linked together in the same order and functionality as HIV or SIV TAT, and then trimerized to generate an immune therapeutic for treating cancer that is well-tolerated and has minimized autoreactivity.

2- The immunotherapeutic of claim 1 that is any non-natural composition (CATS), as defined by less than 95% homology to any HIV-1 or SIV-1 isolate, with tiles identified in this filing (a, b, d, and e as directly below) linked together in order functioning as: a. a signal transduction peptide (STP) at an amino terminus; b. a C-rich determinant (CRD) having homologous human genetic sequences with immunomodulatory ligand activity; c. a (TAR-MTS); d. a loop with acid protease cleavage site(s); and e. a carboxyl trimerization sequence dependent on divalent cation. the resultant trimeric CATS being an immune therapeutic for treating cancer that is humanized so as to reduce its autoreactivity.

3- The humanized trimeric CATS of claim 1 wherein human sequences with identical functionality as the tiles of HIV or SIV Tat are swapped in to replace these sequences with fully human sequences.

4- Claim 3 where human counterpart sequences are identified by a novel algorithm matching anchors in order at the two ends of the functional sequence without giving weight in the match to the intervening sequence.

5- The method of claim 1 where a tile from an SIV Tat lacking pathogenicity is derivatized into the CATS in coordination with other derivatizations.

6- The method of claim 4 where the STP of Tat is replaced by the STP of human wnt-1.

7- The method of claim 4 where the STP of wnt-1 or Tat is modified so as to replace Proline in this SH3 binding domain with Valine.

8- The method of claim 4 where the STP has a WNT-1 like SH3B sequence which is inactivated by replacing all internal Prolines with an amino acid from a group comprising Alanine, Leucine, Isoleucine, Valine, Glycine, and combinations thereof.

9- The method of claim 5 where the STP is derived from SIV Tat so as to activate rather than suppress the immune system.

10- The method of claim 1 where the STP is derived from human CRK and is an activator of the immune system.

11- The method of claim 1 where the immunomodulatory ligand activity is a cysteine-rich ligand for monocyte cells of the innate immune system (CATS).

12- The method of claim 5 where the CRD region is substituted with a natural variant, fully-paired CRD that functions as a ligand-like immunomodulatory for all stages of cancer.

13- The method of claim 5 where the CRD region has 6 Cysteine residues (SEQ ID).

14- The method of claim 5 where the CRD region has 8 Cysteine residues.

15- The method of claim 4 where the CRD contains the CRD from tumor necrosis factor receptor 1 or 2 (TNFR).

16- The method of claim 4 where the CRD contains the CRD from wnt-1.

17- The method of claim 4 where the CRD contains the CRD from TNFR and wnt-1 in tandem.

18- Monoclonal antibodies directed against TNFR used to treat cancer.

19- Monoclonal antibodies directed against wnt-1 used to treat cancer.

20- Bivalent monoclonal antibodies directed against TNFR and wnt-1 used to treat cancer.

21- The method of claim 2 where the divalent-cation dependent trimerization sequence is replaced with an isoleucine or leucine trimerization zipper.

22- The method of claim 21 where the isoleucine zipper is from human TNF-related apoptosis-inducing ligand (TRAIL).

23- The method of claim 21 where the leucine zipper is from human tenascin.

24- The method of claim 21 where the human analogue is ATF-4 (SEQ ID NO.).

25- The method of claim 21 where the trimerization domain is from human collagen XVIII.

26- The method of claim 21 where the trimerization domain is a modified GCN4.

27- The method of claim 21 where each Serine or Threonine residue in the loop region is replaced with an amino acid from a group comprising Alanine, Leucine, Isoleucine, Valine, Glycine, and combinations thereof to provide stabilization.

28- The method of claim 2 where the loop region of Tat is used as a handle or polylinker directly before the trimerization domain to promote trimerization of the whole CATS protein.

Description

DESCRIPTION OF THE FIGURES

[0015] FIG. 1. Schematic Depicting five Tiles Of HIV TAT (SEQ ID NO. 1) and their derivatization into a Humanized CATS Immunotherapeutic with Enhanced Stability.

[0016] From its amino terminus (aa 1-19) Tat encodes a transduction peptide, either an acidic activator or SH3 binding domain (SH3B), followed by a CRD with immunomodulatory ligand activity (aa20-38), followed by the well known TAR-MTS sequence (39-57), followed by a loop with acid protease cleavage sites (58-72), ending with a divalent-cation dependent trimerization sequence. These sequences can be modified individually or in groups to create humanized CATS that are better tolerated and more efficacious than TIRX. Position designations refer to HIV-1 TAT (SEQ ID NO. 1).

[0017] FIG. 2. Graphic depicting the CRD region Of TAT.

[0018] Above. Cn3D view, CRD highlighted in light yellow. Below. Linear schematic, CRD light blue.

[0019] FIG. 3. CRD as the Active site of CATS.

[0020] BALB/c mice implanted with 110.sup.4 4T1 tumor cells were treated s.c. on days 0, day 7, day 14 and day 21with (A) CATS derivatives (400ng) or (B) a TAT derivative with defensin 4 (SEQ ID NO. 9) swapped to replace TAT CRD (SEQ ID NO. 1) (400ng and 2ug s.c.); the control group was treated with PBS. At either dose a defensin 4 CRD (SEQ ID NO. 9) is completely inactive against 4T1 breast cancer.

[0021] FIG. 4. Homology between TAT CRD (SEQ ID NO. 1) and the WNT superfamily of differentiation proteins. (SEQ ID NOS. 10, 11, 11, 12, 13, 14) Alignment structured with the MultAlin tool (INRA).

[0022] FIG. 5. Homology between TAT CRD (SEQ ID NO. 1) and TNF receptor 1 (SEQ ID NO. 15).

[0023] Illustrated is the exact match of the dual C-rich regions spanning respectively only 5 or 6 amino acids when TAT is compared against bat (SEQ ID NO. 15) as detected by the BlastP program.

[0024] FIG. 6. Comparative anti-tumor activity of TIRX versus a prototype humanized CATS in the TS/A breast cancer model.

[0025] Mice (ten per cohort) were implanted s.c. on Day 0 with 110.sup.5 TS/A breast cancer cells and treated weekly starting on Day 1 with 10 ng IV inactive protein (Control, Blue), TIRX (Magenta) or CATS (Green). 1.sup.0 tumor volume is recorded. The difference in 1.sup.0 growth suppression by CATS over TIRX is highly statistically significant (P <0.01).

[0026] FIG. 7. Graph Showing Tumor Immunomodulatory Activity Of CATS Resident In Trimer.

[0027] Trimeric Tat immunotherapeutics (TIRX) but not monomer Tat suppress 1.sup.0 tumor growth of 4T1 murine breast cancer at ng dosing in vivo. A. Western blot of recombinant Tat proteins synthesized in baculovirus after incubation in 10 mM EDTA (Lane 1), 1 mM EDTA (Lane 2),or as directly isolated and purified from insect cells (Lanes 3) probed with polyclonal antibodies to HIV1 SF2 peptide. B. 30 BALB/c mice were implanted in the mammary pad on Day 0 with 110.sup.4 4T1 cells. Beginning on Day 1 and weekly thereafter, ten mice per cohort were treated with either trimcric TAT (TIRX 10 ng IV, purple) which multimerizes spontaneously in baculovirus, standard of care cyclophosphamide (CY 80mg/kg intraperitoneally (IP), green), or monomeric Tat (M-Tat 10 ng IV, brown) produced by incubating TIRX in 10 mM EDTA. Data represent mean tumor volume as calculated (length (mm)width(mm).sup.2)0.52); bars, SE. The difference between CY vs TIRX treatment groups is very highly statistically significant (p<0.005).

[0028] FIG. 8. Schematic Of The Loop And Tail Regions Of Tat.

[0029] Above. Left. Loop region, highlight red. Right. Carboxyl tail, highlighted green. Cn3D model. Below. Linear representation of the loop (red) and tail (blue).

[0030] FIG. 9. Tail Amino acids (66-101) of TAT mediate spontaneous trimerization in Insect Cells. Baculovirus constructs expressing full length, 2 exon TAT, or a construct truncated after aa 65 were used to infect Sf9 insect cells. Proteins were harvested and resolved by SDS-PAGE gel electrophoresis, and probed by immunoblot with polyclonal anti-TAT antibodies. Trimeric TAT resolves at 45 kd, and monomeric TAT runs as a 16 kD protein.

[0031] FIG. 10. SH3 Binding Domain in Pathogenic HIV-1 TAT (SEQ ID NO. 1) has homologies to WNT-1 (SEQ ID NOS. 10, 11, 11, 12, 13, 14).

[0032] A. Cn3D depiction (yellow) and block figure (light blue) of NH.sub.2 signal transduction peptide (STP). B. Praline alignment of HIV-1 (SEQ ID NO. 1), HIV-2 (SEQ ID NO. 16), and CPZ TAT (SEQ ID NO. 17) have perfect alignment of proline residues, while other intervening sequences are more variable. C. MultAlin of HIV-1 TAT (SEQ ID NO. 1) and WNT-1 (SEQ ID NO. 10) demonstrates alignment with two deletions in the viral STP.

[0033] FIG. 11. Block Depiction of a fully-humanized CATS.

[0034] Some or all of these components can comprise any individual CATS compound designed for enhanced safety, production, stability, and efficacy.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Immunotherapy is a targeted approach that could control tumor growth and prevent metastases while avoiding many of the side effects associated with standard chemotherapy. This is particularly relevant in breast cancer where early breast cancer immunotherapy has focused on targeting the immune response against cancer cells with the administration of a vaccine or monoclonal antibody for a breast cancer antigen.

[0036] MAb humanization for therapeutic use is routinely performed prior to clinical trials. Unfortunately the problem of autoreactive toxicity to cancer immunotherapeutics that further stimulate any immune response has not been adequately addressed in the prior art. Their risk of triggering severe autoimmune reactions such as cytokine storms has been reported in several recent cancer clinical trials. The method of the present invention provides a simple platform for mitigating risks at later stage clinical trial. The innovation of this program is to use the humanization steps routinely used in MAb development, in the development of biologic oncoimmunologic (O-I) agonist. The present invention describes a biologic designed for reduced auto-reactivity that can translate safely and efficaciously from animal trials into human clinical trial.

[0037] One embodiment of the present invention anticipates and eliminates auto-reactivity that sensitizes against sustained treatments. This strategy aims to avert failure at costly late-stage clinical trials. For example, Dynavax' TLR adjuvants for hepatitis B vaccines, tested initially in mice, were given to humans at higher doses (Jason D. Marshall, Edith M. Hessel, Josh Gregorio, Christina Abbate, Priscilla Yee, Mabel Chu, Gary Van Nest, Robert L. Coffman, and Karen L. Fearon. Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells. Nucleic Acids Res. 31: 5122-5133, 2003). Dynavax Phase III trial experienced a costly hold when a vaccine developed Wegener's granulomatosis presumed to be a reaction against adjuvant (Hurtado PR, Jeffs L, Nitschke J, Patel M, Sarvestani G, Cassidy J, Hissaria P, Gillis D, Peh CA. CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis. BMC Immunol. 9:34, 2008). While humanization of monoclonal antibody (MAb) therapeutics (Maher VE, Drukman Si, Kinders RJ, Hunter RE, Jennings J, Brigham C,

[0038] Stevens S, Griffin TW. Human antibody response to the intravenous and intraperitoneal administration of the F(ab)2 fragment of the OC125 murine monoclonal antibody. J Immunother 1:56-66, 1992) is now standard, the problem of autoreactive toxicity in cancer immunotherapeutics is compounded by their counter suppressive nature, as evidenced by severe autoimmune reactions (cytokine storms) in recent cancer clinical trials (Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 18:843-851,2010; Voskens CJ, Goldinger SM, Loquai C, Robert C, Kaehler KC, Berking C, Bergmann T, Bockmeyer CL, Eigentler T, Fluck M, Garbe C, Gutzmer R, Grabbe S, Hauschild A, Hein R, Hundorfean G, Justich A, Keller U, Klein C, Mateus C, Mohr P, Paetzold S, Satzger I, Schadendorf

[0039] D, Schlaeppi M, Schuler G, Schuler-Thurner B, Trefzer U, Ulrich J, Vaubel J, von Moos R, Weder P, Wilhelm T, Goppner D, Dummer R, Heinzerling LM. The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network. PLoS One. 8:e53745, 2013).

[0040] One embodiment of the present invention converts a novel C-Rich domain (CRD) that functions as a ligand-like immunomodulator for all stages of murine breast cancer into a safe and sustained therapy for recurrent breast cancers. This is accomplished by swapping in natural variants of the TAT CRD with either 6 C residues (SEQ ID NO. 18), as has been described in Indian HIV Clade C variants with little neurotoxicity (Ranga et. al. JOURNAL OF VIROLOGY, March 2004, p. 2586-2590) or with 8 C residues (SEQ ID NO. 19), as described in SIV-AGM3 which has minimal immunopathology or neurotoxicity (Broussard et. al., Simian Immunodeficiency Virus Replicates to High Levels in Naturally Infected African Green Monkeys without Inducing Immunologic or Neurologic Disease, J. Virol. 2001, 75:2262.) These natural variants with low toxicity replace the toxic HIV TAT CRD (SEQ ID NO. 1) containing an odd 7 C with a fully paired (6 or 8) CRD. Cysteine pairing is a conserved property of natural human CRD. Additionally, sequences derived from human TNFR, from human WNT-1, or both, identified by homology search, can be swapped into the CRD and evaluated for efficacy and safety.

[0041] One embodiment incorporates trimerization strategies to replace the divalent-cation dependent trimerization sequences of TAT with isoleucine or leucine zippers that are less immunogenic, as derivatized from human sequences, and more stable, as lacking cleavage sites. These strategies make for better stability of the product and higher yields of trimer. Trimeric TAT ImmunoTherapeutics (TIRX) deliver, highly statistically significant (p<0.01) reductions in primary tumor mass (50% on average), diminution of pulmonary metastases (approximately 80-90% dependent on study protocols), and survival benefits surpassing by at least two fold paclitaxel standard of care in advanced murine orthotopic 4T1 breast cancer However, TIRX are a potent antigen rapidly inducing self-directed antibodies that neutralize anti-breast cancer activity, limit response time and strength, and occasionally trigger autoreactive toxicities including sudden deaths bearing marked similarities with cytokine storms associated with other cancer immunotherapeutics.

[0042] One embodiment inactivates WNT-1 like SH3B sequences at the amino terminus of TAT by replacing internal P residues with valine. This embodiment either alone, or in combination with substitutions in the loop region that block the generation of monomer, such as replacement of cleavable S and T residues with A, or substitution of the loop with a polyG spacer, negate activity of the SH3B domain. A preferred embodiment of this invention, described previously for DAGRs, replaces the inhibitory WNT-1 sequences with immunostimulatory sequences from the CRK oncogene (SEQ ID NO. 8).

[0043] FIG. 1. Schematic Depicting five Tiles of HIV TAT and their derivatization into a Humanized CATS immunotherapeutic with Enhanced Stability.

[0044] The native HIV TAT protein (SEQ ID NO. 1) was broken down into 5 distinct activities through extensive homology searches, deep data mining, and functional swap derivatizations. At its amino terminus TAT encodes a transduction peptide, typically an acidic activator. At some point during its evolution SIV incorporated an SH3 binding (SH3B) domain instead of an acidic activator, at the same time it became more virulent. The SH3B is found only in HIV-1 (SEQ ID NO. 1), some HIV-2 (SEQ ID NO. 16), and CPZ (SEQ ID NO. 17), all viruses that promote a rapid course to AIDS. As for any SH3B, this function can be inactivated through the preferred conversion of internal P to V.

[0045] TAT's CRD bears strong homology at its amino half to the two C-flanking regions of TNFR1 (SEQ ID NO. 15), and at its COOH half to WNT-1 (SEQ ID NO. 10). Interestingly the C-flanking regions of TAT and bat TNFR1 bear perfect homology. Either or both human peptides can be engineered to replace TAT's CRD, or MAb against the two, including bivalent MAb, could be generated in an attempt to trigger the anti-cancer O-I activity of TAT.

[0046] The cleavage loop that facilitates transition of trimeric TAT to monomer TAT can be stabilized with the replacement of each cleavable S or T residue with A. Alternately, it can be removed entirely and replace with a poly G linker in the context of a leucine or isoleucine trimerization domain. This latter strategy has the advantage that a highly immunogenic sequence of TAT, as measured by its mutability, is replaced by a markedly less immunogenic sequence. The second exon of HIV TAT, as demonstrated in FIG. 9, encodes sequences necessary for trimerization. These sequences, like the cleavage loop, are highly immunogenic as demonstrated by mutability from immune pressure. Replacement with a human isoleucine or leucine trimerization domain has the duel advantages of stabilizing the CATS trimer, and of humanizing a previously autoreactive domain.

[0047] FIG. 2. Graphic depicting the CRD region of TAT.

[0048] FIG. 3. CRD as the Active site of CATS.

[0049] Comparison between CATS and a TAT derivative bearing a defensin 4 CRD (SEQ ID NO. 9). The more active CATS also has a modified SH3B in which internal P are replaced with V. Data represents mean tumor volume calculated as (length (mm)width (mm).sup.2)0.52; bars, SE. Each group contained 10 mice. From day 15 the differences between the control group and groups treated with CAT2 or CAT1 was significant (P <0.05*). The differences between Control and CAT2 or CAT1 treatment groups were highly significant (P <0.01**) starting at day 22. There is no statistical difference between construct with defensin 4 CRD (SEQ ID NO. 9) (low and high dose) and controls.

[0050] FIG. 4. Homology between TAT CRD and the WNT superfamily of differentiation proteins. (SEQ ID NOS. 10, 11, 11, 12, 13, 14) The alignment is focused toward the COOH side of TAT's CRD.

[0051] FIG. 5. Homology between TAT CRD and TNF receptor 1.

[0052] The homology is focused into the NH.sub.2 region of TAT's CRD. TAT has completely deleted all intervening sequences between the C-rich stretches of TNFR1 (SEQ ID NO. 15). The identity with bat TNFR1 (SEQ ID NO. 15) is 9/9 amino acids, while the identity with human TNFR1(SEQ ID NO. 15) is 6/9 amino acids, while still completely matched at the two paired C residues. The Human-TAT CRD homology is insufficient to be detected by BlastP, or for that matter by Clustal, and the standard EMBOSS program.

[0053] FIG. 6. Comparative antitumor activity of TIRX versus a prototype humanized CATS in the TS/A breast cancer model.

[0054] A prototype CATs engineered with a modified CRD and an inactivated SH3B has been preliminarily evaluated in the TS/A murine breast cancer model (FIG. 6). Mice orthotopically implanted on Day 0 in the mammary fat pad were administered starting at Day 1 biweekly intravenous injections of 10 ng of inactive control construct (FIG. 6, Control, blue), TIRX (FIG. 6, magenta) or CATS (FIG. 6, orange) and followed for primary tumor mass (FIG. 6) and survival (data not shown). Tumor progressed rapidly in control animals, which were pre-morbid by Day 30. Animals receiving TIRX partially responded to the first four TIRX doses, but after Day 15 further TIRX administration was ineffective since tumor resumed rapid growth (FIG. 6), resulting in death approximately ten days later than controls (not shown). In contrast, animals receiving biweekly CATS exhibited sustained tumor arrest through Day 17 (FIG. 6), after which 4 of 10 animals progressed, albeit at a significantly slower rate, while the remaining six mice remained in remission throughout a five-week course of therapy spanning 10 doses, at which point the trial was terminated because all control mice had died.

[0055] FIG. 7. Graph showing tumor immunomodulatory activity of CATS resident in Trimer.

[0056] As seen in FIG. 7, Tat spontaneously forms trimers in baculovirus-infected insect cells. Such cysteine rich multimers possess immunomodulatory activity (Jongrak Kittiworakarn, Alain Lecoq, Gervaise Moine, Robert Thai, Evelyne Lajeunesse, Pascal Drevet, Claude Vidaud, Andre Menez, and Michel Leonetti, HIV-1 Tat Raises an Adjuvant-free Humoral Immune Response Controlled by Its Core Region and Its Ability to Form Cysteine-mediated Oligomers, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, 3105-3115, 2006). Stable non-disulfide linked trimeric derivatives such as CRD-containing CATS are immunomodulatory against murine breast cancer in vivo at picomolar dosing (10 ng). At an identical 10 ng dose, monomeric Tat made from the same baculovirus vector after treatment of protein in 10 mM EDTA is inactive as a therapeutic in animals bearing murine breast cancers. At these concentrations monomeric Tat is inactive as well as an extracellular transactivator of the HIV ltr in T cell lines, which typically requires concentrations of 1-10 g/ml.

[0057] FIG. 8. Schematic of the Loop and Tail Regions of Tat.

[0058] The linked loop and tail structures of TAT together promote divalent-cation dependent trimerization, and conversion of the trimer to monomer through acid protease cleavage sites (S and T) typically found in the loop approximately at amino acid 65.

[0059] FIG. 9. Tail Amino acids (66-101) of TAT mediate spontaneous trimerization in Insect Cells. Tat loops typically contain several acid protease cleavage sites clustered between aa62-65. Cleavage at this site is mimicked by the 1 exon construct. In the cell, acid protease cleavage in the lysosome would be expected to transition trimeric TAT to monomer, and thereby expose TAT's STP among other sequences. FIG. 10. SH3 Binding Domain in Pathogenic HIV-1 TAT has homologies to WNT-1. The alignment of TAT to WNT-1 (SEQ ID NO. 10) in the STP complements the downstream alignment to part of the CRD. Emerging is a pattern where two anchors at either end of an alignment between viral and human genes is interspaced by differing sequences or deletions. Current computer algorithms are ill-equipped to detect these alignments. The dually anchored alignment of TAT and WNT-1 (SEQ ID NO. 10) strengthens the credibility of either.

[0060] FIG. 11. Block Depiction of a fully-humanized CATS.

[0061] In order is projected an inactivated SH3B with P switched to V, an attenuated CRD with either 6 or 8 C instead of 7 as contained in canonical virulent TATs, TAR/MTS sequence which has no described toxicities, a transition sequence that replaces aa 65-72 in the loop with a non-cleavable poly G linker, and one of several leucine or isoleucine zippers described in this invention that mediate trimerization of E coli lysates.

[0062] Although the present invention has been described with reference to specific embodiments, workers skilled in the art will recognize that many variations may be made therefrom and it is to be understood and appreciated that the disclosures in accordance with the invention show only some preferred embodiments and advantages of the invention without departing from the broader scope and spirit of the invention. It is to be understood and appreciated that these discoveries in accordance with this invention are only those which are illustrated of the many additional potential applications that may be envisioned by one of ordinary skill in the art, and thus are not in any way intended to be limiting of the invention. Accordingly, other objects and advantages of the invention will be apparent to those skilled in the art from the detailed description together with the claims.'

TABLE-US-00001 SEQ ID No Peptidesequence Source 1 MEPVDPNLEPWKHPGSQPRTACTNCYCKKCCFHCQVCFIRKGLGISYGRK Human KRRQRRRAPQDSQTHQASLSKQPASQSRGDPTGPTESKKKVERETETDPF HIV-1TAT 2 MGAMTQLLAGVFLAFLALATEGGVLKKVIRHKRQSGVNATLPEENQPVVF Humantenascin NHVYNIKLPVGSQCSVDLESASGEKDLAPPSEPSESFQEHTVDGENQIVF precursor THRINIPRRACGCAAAPDVKELLSRLEELENLVSSLREQCTAGAGCCLQP ATGRLDTRPFCSGRGNFSTEGCGCVCEPGWKGPNCSEPECPGNCHLRGRC IDGQCICDDGFTGEDCSQLACPSDCNDQGKCVNGVCICFEGYAGADCSRE ICPVPCSEEHGTCVDGLCVCHDGFAGDDCNKPLCLNNCYNRGRCVENECV CDEGFTGEDCSELICPNDCFDRGRCINGTCYCEEGFTGEDCGKPTCPHAC HTQGRCEEGQCVCDEGFAGVDCSEKRCPADCHNRGRCVDGRCECDDGFTG ADCGELKCPNGCSGHGRCVNGQCVCDEGYTGEDCSQLRCPNDCHSRGRCV EGKCVCEQGFKGYDCSDMSCPNDCHQHGRCVNGMCVCDDGYTGEDCRDRQ CPRDCSNRGLCVDGQCVCEDGFTGPDCAELSCPNDCHGQGRCVNGQCVCH EGFMGKDCKEQRCPSDCHGQGRCVDGQCICHEGFTGLDCGQHSCPSDCNN LGQCVSGRCICNEGYSGEDCSEVSPPKDLVVTEVTEETVNLAWDNEMRVT EYLVVYTPTHEGGLEMQFRVPGDQTSTIIQELEPGVEYFIRVFAILENKK SIPVSARVATYLPAPEGLKFKSIKETSVEVEWDPLDIAFETWEIIFRNMN KEDEGEITKSLRRPETSYRQTGLAPGQEYEISLHIVKNNTRGPGLKRVTT TRLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTID LTEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTTGLDAPRNLR RVSQTDNSITLEWRNGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKT TLTGLRPGTEYGIGVSAVKEDKESNPATINAATELDTPKDLQVSETAETS LTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYN VLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRLNWTAADQA YEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQG YRTPVLSAEASTGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQ EADTVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVE VLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAH NLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTEDLPQL GDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLR AVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVSDIT PESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAHISGLPP STDFIVYLSGLAPSIRTKTISATATTEALPLLENLTISDINPYGFTVSWM ASENAFDSFLVTVVDSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVS GFTQGHQTKPLRAEIVTEAEPEVDNLLVSDATPDGFRLSWTADEGVFDNF VLKIRDTKKQSEPLEITLLAPERTRDITGLREATEYEIELYGISKGRRSQ TVSAIATTAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGG TPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTALD GPSGLVTANITDSEALARWQPAIATVDSYVISYTGEKVPEITRTVSGNTV EYALTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDLDSPRDLTATEVQS ETALLTWRPPRASVTGYLLVYESVDGTVKEVIVGPDTTSYSLADLSPSTH YTAKIQALNGPLRSNMIQTIFTTIGLLYPFPKDCSQAMLNGDTTSGLYTI YLNGDKAEALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDR REEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYK LKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCH RVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGRRKR 3 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYS Human KSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETI TNF-related STVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRK apoptosis- INSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENT inducingligand KNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFEL (TRAIL) KENDRIFVSVTNEHLIDMDHEASFFGAFLVG 4 SSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEAR HumanCollagen TPLPRGTDNEVAALQPP XVIIITrimer Domain 5 MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWL Saccharomyces FCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPC cerevisiae VNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLT GCN4 NKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS 6 MTEMSFLSSEVLVGDLMSPFDQSGLGAEESLGLLDDYLEV Human AKHFKPHGFSSDKAKAGSSEWLAVDGLVSPSNNSKEDAFS ATF-4 GTDWMLEKMDLKEFDLDALLGIDDLETMPDDLLTTLDDTC DLFAPLVQETNKQPPQTVNPIGHLPESLTKPDQVAPFTFL QPLPLSPGVLSSTPDHSFSLELGSEVDITEGDRKPDYTAY VAMIPQCIKEEDTPSDNDSGICMSPESYLGSPQHSPSTRG SPNRSLPSPGVLCGSARPKPYDPPGEKMVAAKVKGEKLDK KLKKMEQNKTAATRYRQKKRAEQEALTGECKELEKKNEAL KERADSLAKEIQYLKDLIEEVRKARGKKRVP 7 MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWL Human FCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPC GITRL VNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLT NKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS 8 MAGNFDSEERSSWYWGRLSRQEAVALLQGQRHGVFLVRDS Human STSPGDYVLSVSENSRVSHYIINSSGPRPPVPPSPAQPPP CRK GVSPSRLRIGDQEFDSLPALLEFYKIHYLDITTLIEPVSR SRQGSGVILRQEEAEYVRALFDFNGNDEEDLPFKKGDILR IRDKPEEQWWNAEDSEGKRGMIPVPYVEKYRPASASVSAL IGGNQEGSHPQPLGGPEPGPYAQPSVNTPLPNLQNGPIYA RVIQKRVPNAYDKTALALEVGELVKVTKINVSGQWEGECN GKRGHFPFTHVRLLDQQNPDEDFS 9 MRVLYLLFSFLFIFLMPLPGVFGGIGDPVTCLKSGAICHPVFCPRRYKQI Human GTCGLPGTKCCKKP -defensin4 10 mglwallpgwvsatlllalaalpaalaanssgrwwgivnvasstnlltds Human kslqlvlepslqllsrkqrrlirqnpgilhsvsgglqsavreckwqfrnr WNT-1 rwncptapgphlfgkivnrgcretafifaitsagvthsvarscsegsies ctcdyrrrgpggpdwhwggcsdnidfgrlfgrefvdsgekgrdlrflmnl hnneagrttvfsemrqeckchgmsgsctvrtcwmrlptlravgdvlrdrf dgasrvlygnrgsnrasraellrlepedpahkppsphdlvyfekspnfct ysgrlgtagtagracnssspaldgcellccgrghrtrtqrvtercnctfh wcchvscrncthtrvlhecl 11 maplgyflllcslkqalgsypiwwslavgpqysslgsqpilcasipgivp Human kqlrfcrnyveimpsvaegikigiqecqhqfrgrrwncttvhdslaifgp WNT-3a vldkatresafvhaiasagvafavtrscaegtaaicgcssrhqgspgkgw kwggcsediefggmvsrefadarenrpdarsamnrhnneagrqaiashmh lkckchglsgscevktcwwsqpdfraigdflkdkydsasemvvekhresr gwvetlrprytyfkvpterdlvyyeaspnfcepnpetgsfgtrdrtcnvs shgidgcdllccgrghnaraerrrekcrcvfhwccyvscqectrvydvht ckpchswatgregrrrwstlgcgprdgclrtghsgpcrslawiwspgsqg hdlleqlprsgglgqcsslqnwtavsgclrdhlgglpgggehgdts 12 CNCKFHWCCYVKCNTCSEI Human WNT-1(c) 13 MLEEPRPRPPPSGLAGLLFLALCSRALSNEILGLKLPGEPPLTANTVCLT Human LSGLSKRQLGLCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGG WNT-10b GRLPHHSAILKRGFRESAFSFSMLAAGVMHAVATACSLGKLVSCGCGWKG SGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPSPGPQDTWEWGGCN HDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVTENLKRKCKCH GTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLR PRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCG RGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCK 14 CNCKFHWCCAVRCEQCRRI Human WNT-1(e) 15 MMSRSGSGEEDSHTWTYRYNDCPAPGRDTYCKKCENGTYTASENYLSQCI TNFreceptor1A SCSICRKEMGQVEISPCTVDQNTVCGCKKNQYQESLSDTLFRCRNCSPCL (myotisbrandtii) NGTVQISCSAKQNTVCTCHTGFFLKDNKCVPCDNCEKNTECTKLCPSTGE VIGGSPDSVLLSLVIFFGFCLLCLLFMGLTCHFQRWKPKLQSIGGAGAPA LRPRLQPHHRLQSHPQLHAKFHLYPW 16 METPLKAPESSLKPYNEPSSCTSERDVTAQELAKQGEELLAQLHRPLEPC HIV-2 TNKCYCKRCSFHCQLCFSKKGLGISYERKGRRRRTPRKTKTPSPSAPDKS ISTRTGDSQPTKRQKKTSEATVVTTCGLGQ 17 MDPIDPDLEPWKHPGSQPRTVCNNCYCKACCYHCIYCFTKKGLGISYGRK CPZTAT KRTTRRRTAPAGSKNNQDSIPKQPLSQSRGNKEGSEKSTKEVASKTEADQ 18 KTACNNCYCKHCSYHCLVCFQKKGLG 6CysteineCRD 19 KRCTNKCYCKCCCYHCQLCFLQKGLG 8CysteineCRD