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Transcript
United States Patent [191 [11] 4,107,298 Liining [45] Aug. 15, 1978 [54] ANTIGENICALLY ACTIVE POLYPEPTIDE 4,018,753 AND A PROCESS FOR ITS PREPARATION [75] Inventor: Bj'tirn Erik Gustav Bertilsson Liining, Stockholm, Sweden [73] Assignee: AB Bonnierforetagen, Stockholm, Sweden 4/1977 Inouye et a1. ............... .. 260/1125 R FOREIGN PATENT DOCUMENTS 787,918 728,129 6/1968 2/1966 Canada ........................... .. 260/1125 R Canada ........................... .. 260/1125 R Primary Examiner-Delbert R. Phillips Attorney, Agent, or Firm—-Gordon W. Hueschen [21] Appl. No.: 789,665 [57] [22] Filed: Apr. 21, 1977 [30] Foreign Application Priority Data The invention provides for a synthetic antigenically active polypeptide containing an immunologically inert Apr. 29, 1976 [SE] [51] ABSTRACT portion and as an active immunological determinant Sweden .............................. .. 7604972 Int. Cl.2 ................... .. A61K 37/00; C07C 103/52 group the amino acid sequence A_3-A_2-A_1-A0-A1 A2-A3, wherein A0 is an arginine residue, A1, A2, A_l [52] US. Cl. . . . . . . 424/177; 260/ 112.5 R and A_2 are the same or different and selected from [58] Field of Search ................ .. 260/ 1 12.5 R; 424/ 177 residues of: glycine, alanine, valine, leucine, lysine, and [56] . . . . . . . . . . . . .. isoleucine and A3 and A_, are same or different and References Cited selected from glutamic acid and aspartic acid. The in U.S. PATENT DOCUMENTS 3,715,190 2/1973 3,929,756 3,960,827 12/1975 6/1976 Won Kil Park et al. vention also provides for a process for the preparation 260/1125 R Leeman et al. ..... .. 260/1125 R Bjorklund .......................... .. 424/177 of such polypeptide. 12 Claims, 1 Drawing Figure 1 4,107,298 2 wherein A4 and A_., are the same or different and se lected from residues originating from glycine, alanine, ANTIGENICALLY ACTIVE POLYPEPTIDE AND A PROCESS FOR ITS PREPARATION valine, leucine and isoleucine which, as indicated above, are neutral aminoacids. In view of thecharacter of the aminoacids in said respect, the fractions of the aminoa The present invention relates to a synthetic antigeni cally active polypeptide which is characterized by con taining an immunologically inert portion and as an im munologically determinant group, a certain aminoacid sequence, and a process for the preparation of said poly peptide. ' cid sequence flanking the centrally positioned arginine residue indicated A0 ‘are still‘, mirror-images of each other. Other symbols in this preferred aminoacid se quence have the meanings given above. 10 (III) According to a particularly preferred embodi In Swedish Pat. application 73-08917-9 and in US. Pat. No. 3,960,827 there is described a cancer-associated ment of the present invention, a tyrosine residue is at tached to the aminoacid residue A_4, said tyrosine resi polypeptide antigen (CAPA), the technique for its isola due, as is clear from the following description, playing tion and its use in cancer diagnosis and in the prepara a relatively essential role for the activity of the product. tion of antibodies. The CAPA is now commercialized The sequence A_4 through A0 is most important in this under the designation TPA, which stands for “tissue embodiment. polypeptide antigen”. As is clear from the speci?cation (IV) In the last-mentioned embodiment containing a of the above-identi?ed patent application and US. pa tyrosine residue Al and A4 are preferably alanine resi tent, the isolation of the natural antigen is a complicated dues, A2, A_; and A_., are leucine residues, and A3 a procedure which, even if resulting in a practically use 20 glutamic acid residue, A_, being a valine residue and ful product, still involves high production costs and A_; an aspartic acid residue. moreover may involve dif?culties in the provision of From hereon the following abbreviations for the necessary starting materials, such as tumor tissue, etc. Against this background a synthetically prepared anti aminoacids in question are used: gen would, of course, be very attractive, in view of the 25 possibility of thereby obtaining a product exactly speci ALANINE Ala ?ed as to its composition, said product also being capa ble of being prepared at a more favorable price. Referring to the drawing, the FIGURE shows in the ARGININE Arg ASPARAGINE Asn hemagglutination inhibition test, comparison between the natural CAPA and the representative synthetic antigen of this invention according to Example 1. This FIGURE will be further explained hereinafter. ASPARTIC ACID Asp GLUTAMINE GLUTAMIC ACID GLYCINE ISOLEUCINE vLEUCINE TYROSINE VALINE Gln Glu GLY Ile Leu TI-IREONINE Tyr PHENYLALANINE Val LYSINE Thr Phe Lys The main object of this invention is thus to provide a synthetic antigenically active polypeptide which reacts monospeci?cally with antibodies prepared by means of the polypeptide antigen described in the above-identi ?ed patent application. Using the above abbreviations, in a particularly pre ferred embodiment A5 is an alanine residue, and A6 and A_6are the same or different and selected from residues of glutamine and asparagine. In this aminoacid se According to one aspect of the instant invention, there is thus provided a synthetic antigenically active 40 quence, the following speci?c sequence is particularly preferred: (V) Gln-Tyr-Leu-Asp-Leu-Val-Arg-Ala polypeptide characterized by containing as an active constituent or immunological determinative group the amino acid sequence: (I) A_3-A_2-A_1-A0-Al-A2-A3, wherein A0 indicates an arginine residue A1, A2, A_l Leu-Glu-Ala-Ala-Asn. Moreover, it is preferred in this sequence at both ends thereof to connect as A7 a glycine residue and as A_7 an and A_; are the same or different and are selected from 45 alanine residue, i.e., using the above abbreviations the aminoacid residues derived from glycine, alanine, val ine, leucine, lysine and isoleucine. A; and A_; are the same or different and selected from residues derived from glutamic acid and aspartic acid. sequence: (VI) Ala-Gln-Tyr-Leu-Asp-Leu-Val-Arg Ala-Leu-Glu-Ala-Ala-Asn-Gly. In the foregoing formulas V and VI Gln may be replaced equally well by lysine or Asn, or vice versa. As is clear from the above aminoacid sequence, the The present invention also relates to a process for the two fractions ?anking the centrally positioned arginine preparation of the antigenically active polypeptide, and in this process an N-protected amino acid is attached to a resin by esteri?cation, the N-protecting group is re moved and a second N-protected amino acid is coupled from neutral aminoacids, namely glycine, alanine, val ine, leucine and isoleucine. Moreover, the aminoacid 55 to the amino group of the resin-bound amino acid, the residue indicated A0 are mirror-images of each other, in that the two aminoacid residues adjacent to A0 originate residues indicated by A3 and A_;, respectively, are derived from negative or acidic aminoacids, namely N-protecting group is removed and the coupling step is repeated with a third N-protected amino acid. Protect ing groups in the process are the usual groups, and are set forth more fully in the following. This procedure is atory arginine residue is moreover derived from a posi tive or basic aminoacid, namely arginine, the basicity of 60 repeated until the desired amino acid sequence is ob glutamic acid and aspartic acid, respectively. The oblig which, however, is superseded by the combined acidi ties of the aminoacid residues indicated by A3 and A_;. Thus, the aminoacid fraction imparting the desired anti genic activity to the polypeptide will thus, taken as a tained, the polypeptide being then cleaved from the quence: constituting the major part of the copolymer, for in resin. It is preferred after each attachment of an N protected amino acid to the resin-bound amino acid to wash away all by products and unreacted soluble mate 65 rials. whole, have a negative or acidic character. The resin used in the synthesis may consist of a co According to a preferred embodiment of the instant polymer of styrene and divinyl benzene, the styrene invention, the polypeptide contains the aminoacid se A _4-A_ 3'A_ Z'A _1'A0'A1'A2'A3'A4, 4,107,298 3 dleman, stance about 98% of styrene and about 2% of divinyl agents are also suitable and will be apparent to one skilled in this art. The present invention will now be illustrated further by means of non-limiting examples. In order to provide a reactive group for coupling to the ?rst amino acid, the benzene rings are suitably par tially chloromethylated. When this chloromethylated resin is treated with the triethylamine salt of an N protected amino acid, a bond of the benzyl ester type is formed. Such bond is stable under the synthesis steps, EXAMPLE 1 By the process in solid phase according to Merri?eld but can be cleaved with HBr in acetic acid or tri?uoro (cf. the above literature reference) the following prod acetic acid, the N-protecting group being simulta neously removed and the peptide separated from the R25—R24 . . . . . Berlin-Heidelberg-New York, 1970, particularly pp. 308-310. Other coupling benzene. R26 4 Springer-Verlag, uct is prepared: . R4—R, Rl | Boc-NH-CH-CONH-CH ——————— —— CH-CO- NH=CH—CO—NH-CH—COO—CHZ—@— R wherein: R indicates the resin part; B00 is a t-butyloxy resin. In order to protect the amino acid the t-butyloxycar carbonyl group; CH3 CH3 NH bonyl group can suitably be used as it is conveniently cleaved by means of HCl in acetic acid. It is also possi ble to use the carbobenzoxy group for the purpose, but The amino acid sequence between the t-butyloxycar in this case HBr in acetic acid must be used for removal 55 bonyl group and the resin part may be expressed in the of the protecting group. As a protecting group there following manner using the foregoing abbreviations may also be used the o-nitrophenyl sulfenyl group. regarding the amino acids: Other protecting groups may also be employed and will be apparent to one skilled in the art. The coupling reagent most frequently used in the 60 synthesis is dicyclohexylcarbodiimide. If methylene Ala Leu Leu Asn Asp Glu Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Gln Leu Glu Val (v11) chloride is used as a solvent and about 50% excess of 0.4 mmole of Boc-valine (resinbenzyl ester) was transferred to the cuvette of a Beckman peptide synthe~ diimide, a quantitative reaction is obtained within a few minutes. Even dimethylformamide may be used as a 65 sizer and swollen in methylene chloride (CHZCIZ). The protecting Boc-group was removed by treatment with solvent. As regards further details regarding the synthe tri?uoroacetic acid in excess in methylene chloride. sis procedure reference is had to the book “Protein After washing with methylene chloride the resin was Sequence Determination” summarized by Saul. B. Nee t-butyloxy carbonyl aminoacid and dicyclohexyl carbo 4,107,298 5 6 neutralized with triethylamine and again washed with methylene chloride. N-Boc-glutamic acid benzyl ester (coupling step 1) and cyclohexylcarbodiimide was peptide according to the present invention is of decisive added dissolved in CHZCIZ, and the mixture was stirred for 30 minutes. The resin was washed and the coupling tyrosine designated A_5, the polypeptide was treated step repeated. This terminates the introduction of R2 in step 1 according to the above. Step 2 of the synthesis starts by removal of an N-Boc from the N-terminated amino acid and repetition of the coupling step while using another N-Boc-amino acid. about 10000 times in relation to the tyrosine content of importance for the antigenic activity. For the purpose of investigating the effect of the with iodine at pH 9.5. When using iodine in an excess of the polypeptide, about 85% of the activity is lost, at 1000 times excess about 75% of the activity is lost, whereas at 100 times excess 40-50% of the activity disappears. At an excess of only about 10 times the To build up the above amino acid sequence the follow activity of the polypeptide is, however, not effected. ing reagents are used in the subsequent steps: From the character of the basic structure, it is clear that the polypeptide has an acidic character, thus being negatively charged in a neutral solution while, how in step N-Boc-Leucine I: N-Boc-asparagine-xanthydryl derivative u N-Boc-alanine Do as in step 6 ever, maintaining the positive charge of the arginine part thereof. The chemical basis for the antigenic character of the polypeptide has thus been set forth in the foregoing Do as in step 1 D20 as in step 2 D20 as in step 6 20 been obtained by means of a synthetically prepared D20 as in step l N-Boc-glycine n description. The remarkable speci?c activity which has N-Boc-G-tosylargine product, the activity of which can be said to be of a haptenic nature, due to the small determinant group, constitutes a pioneering advance with regard to the N-Boc-valine Dzo as in step 2 N-Boc-aspartic acid benzyl ester Dzo as in step 2 N~Boc-tyrosine-3-chlorobenzyl ether N-Boc-glutamine~xanthydryl derivative Do D20 Do D20 Dzo Dzo Do D20 as as as as as as as as in in in in in in in in step step step step step step step step 6 2 1 14 5 2 2 6 application of immunology and testing, i.e. diagnosis, 25 within the cancer area. Clearly and in accordance with what is known in immunology, antigenic activity is a function not only of the structure of the haptenic determinant group, but also of the size of the polypeptide. Said size does not, 30 however, effect the speci?city of the peptide but only the degree of activity, inasmuch as a larger molecule tends to be more active than a smaller one. Therefore, an improved activity is obtained if the polypeptide is The protecting groups are removed from the pro tected resin-bond peptide and the resin is cleaved off by arranged on a suitable immunogenically active carrier, using anhydrous hydrogen ?uoride at 0° C for 30 min 35 for instance a protein, such as albumin, for instance egg white. Already the polypeptide in its basic structural utes, and the peptide is extracted from the resin by means of a 10% aqueous solution of acetic acid. After form, i.e., having at least 7 amino acid units, does, how ever, show an antigenic activity which is suf?cient to evaporation the residue is puri?ed by gel ?ltration on enable the polypeptide to react with corresponding Sephadex G 25 Fine in 0.1 M NH4HCO3. After lyophili zation of the peptide a satisfactory amino acid analysis is 40 antibodies. Moreover, the polypeptide may be used for obtained, together with a molecular weight of approxi generating antibodies; however, this calls for coupling the polypeptide to a suitable protein acting as a carrier mately 3000, as determined by gel ?ltration. The theo retical MW value is 2959.71. or so-called “Schlepperantigene,” for instance swine The polypeptide prepared according to the foregoing serum. Thus, the activity is imparted to the polypeptide was investigated with regard to its ability to inhibit the 45 chain beginning at as low as 7 and especially 9 amino hemagglutination reaction between tanned red sheep acid units, appears to be maximized at about 26 amino blood cells labelled with natural cancer antigen and acid units, and is still present at in excess of thirty amino antibodies prepared by using natural cancer antigen acid units, the portion of the polypeptide chain not being an immunological determinant group as hereinbe (CAPA or TPA). With regard to details concerning this fore de?ned being inert immunologically but serving to technique reference is had to the above-identi?ed Swedish patent application 73-08917-9 and US. Pat. provide a “carrier” for the immunologically determi No. 3,960,827. The speci?c activity of the polypeptide nant groups. The present invention is in no way limited to the was found to be 0.024 Units per milligram (U/mg). Said Unit for speci?c activity is de?ned as l/ 6 of the quantity active polypeptide required to label 109 sheep blood cells so as to be fully agglutinated by a minimum num ber or amount of antibodies (maximum dilution of anti foregoing speci?c embodiments. Thus, in preparing the 55 polypeptide, the resin may be any material containing benzene rings and in addition at least one group having the ability of binding N-protected amino acids by esteri ?cation. The ester bond must be relatively easy to hy For the purpose of investigating what functions that drolyse and, of course, must be easier to cleave than the are critical for the activity of the polypeptide, the basic 60 peptide bonds in the polypeptide. However, the ester bond must be suf?ciently stable to be able to withstand structure of which is given above, certain experiments were carried out. The centrally positioned arginine the reaction conditions under the synthesis. indicated by A0 was thus blocked with cyclohexanedi As an alternative to the above mentioned coupling one at pH 13 resulting in 99.5% loss of activity. If, reagent, dicyclohexyl carbodiimide, when attaching however, the polypeptide is subjected only to a basic 65 glutamine and asparagine units, coupling may instead be carried out with so-called active esters, for instance environment, wherein the pH is 13 for the same period cyanomethyl, thiophenyl or nitrophenyl esters. As a of time, the activity is lowered only by about 20%. This indicates the fact that the arginine presence in the poly solvent in the synthesis so-called aprotic solvents are bodies). 7 4,107,298 suitable, particularly solvents that are somewhat hydrophilic or “semipolar.” 8 activity of the synthesized polypeptide is found to be 0.024 Units per milligram of peptide. Regarding useful groups to protect the N-amino EXAMPLE 2 acids, it is noted that t-butyloxy carbonyl groups, wherein one or two methyl groups are replaced by 5 In the same manner as given in Example 1 the follow phenyl, also may be advantageously used. In this con- ing polypeptide is prepared and found to be about nection it can be mentioned that the product obtained in equally immunologically active as the polypeptide of the synthesis is provided with N-protecting groups and Example 1: attached to a resin by esteri?cation, and that it advanta geously can be stored for a long period of time without 10 Th AS Leu AS A A being destroyed. In connection with the use _of the poly- A‘; Ly‘; Tyr L63 A25 L;§ pept1de, the protectmg groups and the resin part may then be removed. In the amino acid sequences given in the present Arg Leu Glu Ala Ala Ala Gly Glu Leu Glu Val L Asp preparation, the terminal groups (after removal of the 15 protecting groups and the resin part) are always amino EXAMPLE 3 group and carboxyl group, respectively. The amino In the same manner as given in Example 1 the follow group is found at the end of the chain wherein the syming polypeptide is prepared and found to be about bols A are designated minus, whereas the carboxyl equally immunologically active as the polypeptide of group is found at the opposite end of the amino acid 20 Example 1: sequence. the antigenic- activ. In Example 1 as. described above . Ala Gln Tyr Leu Asp Leu Val Arg Ala ity of the synthesized polypeptlde was determlned 1n Glu Ala Ala Asn Gly Glu Leu Glu val Leu the following manner. For quantitative determination of the amount of poly- 25 When this determinant group is coupled to inert bo peptide 7 mgs of the peptide are dissolved in 1.4 mls of vine albumin in a molar ratio of about 1:1 by means of a buffer grade serum, ie a physiological saline solution water soluble carbodiimide (EDC) the immunological containing 2% of inert human serum and buffered to a activity of the peptide is maintained. pH of about 7.5 with a phosphate buffer. Under serial dilution a series of samples of said solution having a 30 EXAMPLE 4 decreasing polypeptide concentration is prepared and In the same manner as given in Example 1 the follow to each of said samples there is added a pre-determined amount of anti-serum containing antibodies speci?c to TPA. To each of the resulting samples there is then added, after incubation, a predetermined amount of the Leu Arg ing polypeptide is prepared and found to be about equally immunologically active as the polypeptide of Example 1: Asn Ala Asp Leu Glu Glu Leu Ala Ala Ala Gln Asn Tyr Gly Leu Arg Asp Leu Leu Glu Val Val When coupled to inert albumin in the same way as . . . . . . descrlbed in Example 3, the actlvlty was maintained. p 01 yp e p tide carried on a p articulate carrier, hemagg lutination taking place to an extent corresponding to the amount of available antibodies. In parallel there is pre- EXAMPLE 5 pared a corresponding series of control samples con In the same manner as given in Example 1 the follow taining known decreasing amounts of TPA. The diame 45 ing polypeptide is prepared and found to be about ters of the hemagglutination depositions of the control equally immunologically active as the polypeptide of samples are then measured in their respective cavities Example 1: on a serial dilution test plate, and the values of the diam Asn Arg Asp Ala Ile Leu Leu Glu Ala Ala Gln Ala Tyr Asn Leu Gly Asp Leu Val eters obtained are plotted against the TPA concentraEXAMPLE 6 tions, an S-shaped curve being obtained, the intermedi In the same manner as given in Example 1 the follow ate part of which including an in?exion point as a steep 55 ing polypeptide is prepared and found to be about inclination. The corresponding curve of the diameter of equally immunologically active as the polypeptide of the depositions from the hemagglutination as a function Example 1: of the TPA-concentrations is shown in the diagram of Asp Arg Asp Ala Arg Leu Leu Glu Ala Ala Lys Ala Tyr Asp Leu Gly Asp Lys Val EXAMPLES 7-22. the appended drawing, as curve B. 65 In the same manner as given in Example 1 the follow In the same diagram the corresponding curve for the ing polypeptides are prepared and found to be about synthesized polypeptide is shown as A, and with knowledge of the speci?c activity of the TPA the speci?c equally immunologically active as the polypeptide of Example 1. 4,107,298 9 Ex. 7 Ex. 8 Ex. 9 Ex.l0 Ex.ll Ex.l2 Ex.l3 Ex.l4 Ex.l5 Ex,l6 Ex.l7 Ex.l3 Ex.l9 Ex.20 Ex.2l Ex.22 Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Leu Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp 10 Arg Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Glu Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Arg Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Glu Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Arg Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Glu Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Arg Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Glu Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Arg Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Glu Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Arg Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Glu Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Arg Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Glu Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Arg Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val Glu Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val residues of: glycine, alanine, valine, leucine, and isoleu EXAMPLE 23 In the same manner as given in Example 1 the follow cine and A3 and A_; are same or different and selected from glutamic acid and aspartic acid. ing polypeptide is prepared and found to be about 3. Synthetic antigenically active polypeptide, charac equally immunologically active as the polypeptide of terized by containing an immunologically inert portion Example 1 20 and as an active immunological determinant group the Thr Val Asp Arg Phe Leu Ala Asp Asp Asp Arg Leu Ala Lys Tyr Leu Asp Lys Leu Glu Glu Leu Ala Ala Asp Gly Glu Leu Glu Val Asn Leu Gln amino acid sequence: A_4-A_3-A,2-A_l-AO-Al-A2-A3 A4, wherein A0 is an arginine residue, Al, A2, A_, and EXAMPLE 24 In the same manner as given in Example 1 the follow A“ 2 are same or different and selected from residues of: ing polypeptide is prepared and found to be about glycine, alanine, valine, leucine, lysine, and isoleucine equally immunologically active as the polypeptide of 30 and A3 and A_; are same or different and selected from Example 1 glutamic acid and aspartic acid, and A4 and A_,, are Ala Arg Glu Leu Ala Leu Leu Leu Asn Asn Glu Leu Asp Ala Gln Glu Ala Leu Asn Ala Gly Gln Arg Tyr Leu Leu Glu Asp Val Leu Phe Val Asp same or different and selected from g1 ycine ’ alanine ’ EXAMPLE 25 In the same manner as given in Example 1 the follow ing polypeptide is prepared and found to be about equally immunologically active as the polypeptide of Example 1 valine, leucine, and isoleucine. 4. Synthetic antigenically active polypeptide, charac terized by containing an immunologically inert portion and as an active immunological determinant group the amino acid sequence: A_5-A_4-A_3-A_2-A_FAQ-A, Ala Leu Leu Asn Asp Ile Leu Ala Gln Tyr Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Leu Glu Asp Leu Val Val Various modifications and substitutions of equiva lents may be made in the products, process, conditions, and procedures of the present invention and will be 50 AZ-A3-A4, wherein A_5 is a tyrosine residue and immediately apparent to one skilled in the art, where wherein A0 is an arginine residue, A], A2, A_, and A_2 fore the invention is to be limited only by the full scope are same or different and selected from residues of: of the appended claims, including application of the doctrine of equivalents thereto. glycine, alanine, valine, leucine, lysine, and isoleucine and A3 and A_, are same or different and selected from 55 glutamic acid and aspartic acid, and wherein A4 and 1. Synthetic antigenically active polypeptide, charac A_., are same or different and selected from glycine, What is claimed is: terized by containing an immunologically inert portion and as an active immunological determinant group the alanine, valine, leucine and isoleucine. 5. A polypeptide according to claim 4, characterized in that Al and A4 are alanine residues, A2, A_; and A_4 amino acid sequence: A_3-A_2-A_1-A0-Al-A2-A3, wherein A0 is an arginine residue, A1, A2, A_, and A_; 60 are leucine residues and A3 is a glutamic acid residue, are same or different and selected from residues of: glycine, alanine, valine, leucine, lysine, and isoleucine A_, is a valine residue and A_; is an aspartic acid resi due. and A3 and A_;, are same or different and selected from glutamic acid and aspartic acid. ' 6. Synthetic antigenically active polypeptide, charac terized by containing an immunologically inert portion 2. A polypeptide according to claim 1, characterized 65 and as an active immunological determinant group the in that the amino acid sequence is: A_3-A_2-A_1-A0 amino acid sequence: A_b-A_5-A_,,-Ag_,-A_Z-A_l-Ao ArAz-As, wherein A0 is an arginine residue, A‘, A2, A_, and A_2 are same or different and selected from A,-A2-A3~A4-A5-A6, characterized in that Al and A4 and A5 are alanine residues, A2, A_2 and A_,, are leucine 11 4,107,298 12 10. Synthetic antigenically active polypeptide, char residues and A3is a glutamic acid residue, A_ 1 is a valine residue and A_; is an aspartic acid residue, A0 is an acterized by containing an immunologically inert por arginine residue, A_5 is a tyrosine residue, and A6 and tion and as an active immunological determinant group the amino acid sequence: Ala Leu Leu Asn Asp Glu 5 Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu glutamine, asparagine, and lysine. Ala Ala Asn Gly Glu Leu Glu Val. 7. A polypeptide according to claim 6, wherein A6 A_6 are same or different and selected from residues of 11. Synthetic antigenically active polypeptide, char and A_6 are same or different and selected from residues acterized by containing an immunologically inert por of glutamine and asparagine. tion and as an active immunological determinant group 8. A polypeptide according to claim 6, characterized in that A6 is an asparagine residue and A ,,a glutamine residue. the amino acid sequence: Thr Asp Leu Asp Asp Arg Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asp Gly Glu Leu Glu Val. 9. Synthetic antigenically active polypeptide, charac terized by containing an immunologically inert portion 12. Synthetic antigenically active polypeptide, char and as an active immunological determinant group the acterized by containing an immunologically inert por amino acid sequence: A_7-A_(,-A_5 'A_4'A _ 3'A_ 2"A _1‘Ag-A]-A2"A3-A4'A5-A6'A7, Char acterized in that Al and A4 and A5 are alanine residues, A2, A_2 and A_4 are leucine residues and A3 is a glu tamic acid residue, A_l is a valine residue and A_, is an aspartic acid residue, A0 is an arginine residue, A_5 is a tyrosine residue, A6 is an asparagine residue and A_,, a tion and as an active immunological determinant group the amino acid sequence: Leu Asn Asp A_9 Leu Ala A_6 Tyr Leu Asp A_Z Val Arg Ala Leu Glu Ala Ala Asn Gly A8 Leu Glu Val, wherein A_,, and A8 are same or different and selected from residues of glutamic acid and arginine; A_(,is selected from residues of glutamine and lysine; and A_2 is selected from residues of leucine and lysine. glutamine residue, and wherein A7 is a glycine residue i and A_7 an alanine residue. 25 35 40 45 55 60 65 II‘ II‘ * * UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT N0. DATED : : 4,107,298 August 15, 1978 INVENTOR(S) : Liining It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: \ Col. 5, line 21, Step 11 in the chart; "tosylargine" should read —-tosylarginineCol. 11, line 10; "A 6" (2nd occurrence) should read --A_6--. Signal and Scaled this Sixth Day Of February 1979 lsmu A ttest: RUTH a MASON Arresting Oj?cer DONALD W. BANNER Commissioner of Patents and Trademarks