本發明係關於一種結合於人類CCR1之細胞外區域而抑制由人類CCL15引起之CCR1之活化的單株抗體或該抗體片段。 CCR1亦被稱為CD191、CKR-1、HM145、Macrophage inflammatory protein 1α receptor(MIP1αR)、CMKBR1及SCYAR1等。CCR1為具有7次膜貫通型結構之GPCR，為包含全長355個胺基酸之膜蛋白。 已知於包含CCR1之GPCR中，細胞表面上之GPCR藉由配體之結合而得到活化，從而向該細胞內傳遞該受體依賴性訊號，與此同時，該細胞內之鈣離子濃度上升。其結果該細胞發生細胞遷移、趨化素之產生、基質金屬蛋白酶MMP之產生等。 即，作為CCR1之功能，可列舉以下等功能，即，藉由配體結合於細胞表面上之CCR1而向該細胞內傳遞CCR1依賴性訊號，與此同時，該細胞內之鈣離子濃度上升，結果使該細胞發生細胞遷移、趨化素之產生或MMP之產生等。 作為人類CCR1之配體，例如可列舉：人類CCL3、CCL5、CCL8、CCL14、CCL15、CCL16及CCL23等。作為小鼠CCR1之配體，例如可列舉：小鼠CCL3、CCL5、CCL7及CCL9等。 人類CCL15為C-C趨化素家族所含之配體，包含全長92個胺基酸。已知人類CCL15於蛋白質分解酶之作用下N末端經分解，而成為約68胺基酸之活化型[以下，於本發明中記載為活化人類CCL15或hCCL15(68aa)]，藉此發揮較全長之CCL15(以下，於本發明中記載為全長CCL15)更強力之活性。 若人類CCL15結合於細胞表面上之人類CCR1而使該受體活化，則向該細胞內傳遞CCR1依賴性訊號，而引起磷脂酶C(PLC)之活化、細胞內之鈣離子濃度之上升或nuclear factor-κB(NF-κB)之活化等。其結果該細胞發生細胞遷移等。 作為本發明之抗體，可列舉抑制由人類CCL15引起之人類CCR1活化所伴隨之各種反應的抗體。作為本發明之抗體，具體而言，例如可列舉抑制選自由人類CCL15引起之人類CCR1表現細胞內之CCR1依賴性訊號傳遞、PLC之活化、細胞內鈣離子濃度之上升、NF-κB之活化及CCR1表現細胞之遷移中之至少一種反應的抗體等。其中，作為本發明之抗體，較佳為抑制由人類CCL15所誘發之人類CCR1表現細胞之遷移的抗體。 作為本發明之抗體，較佳為列舉針對由人類CCL15引起之人類CCR1活化所伴隨之上述反應，與僅添加CCL15而未添加抗體之對照組相比實現5%以上、10%以上、20%以上、30%以上、40%以上、50%以上、60%以上、70%以上、80%以上、90%以上抑制的抗體。人類CCL15之濃度可根據測定系統而適當調整，使之成為人類CCL15添加時之上述反應之活性成為最大值之濃度。例如於本案實施例所記載之方法中，於測定CCR1表現細胞之遷移之情形時，CCL15之濃度較佳為1 ng/mL。又，本發明之抗體之濃度亦可根據測定系統而適當調整。例如於本實施例所記載之方法中，於測定CCR1表現細胞之遷移之情形時，作為本發明之抗體濃度，可列舉0.3 μg/mL以上，較佳為1 μg/mL以上，更佳為3 μg/mL以上，最佳為10 μg/mL以上。 本發明中，人類CCL15只要為使CCR1活化者，則可為全長CCL15及活化人類CCL15中之任一種CCL15。 作為人類CCR1表現細胞，只要為表現人類CCR1之細胞，則可為任意之細胞，例如可列舉：人類細胞、人類細胞株及人類CCR1強制表現株等。 作為表現人類CCR1之人類細胞，例如可列舉：嗜中性球、嗜酸性球、嗜鹼性球、單核球、巨噬細胞、樹狀細胞、NK細胞、T細胞、B細胞、未成熟骨髄球(iMC)及骨髄源性抑制細胞(MDSC)等。 作為人類CCR1之細胞外區域，可列舉：包含自人類CCR1之胺基酸序列之N末端起之第1～31號胺基酸序列的N末端區域、包含第97～103號胺基酸序列之細胞外環1區域、包含第172～195號胺基酸序列之細胞外環2區域及包含第266～278號胺基酸序列之細胞外環3區域[Cell 72.3(1993)：415-425]。 作為N末端區域、細胞外環1區域、細胞外環2區域及細胞外環3區域，具體而言分別可列舉序列編號2之胺基酸序列中之第1～31號、第97～103號、第172～195號及第266～278號胺基酸序列。 作為本發明之抗體，只要為結合於上述人類CCR1之細胞外區域之抗體，則可為任意者，較佳為結合於人類CCR1之細胞外環2區域之胺基酸序列中之至少1個胺基酸殘基的抗體。作為該抗體，可列舉結合於序列編號2之胺基酸序列之第172～195號胺基酸序列中之至少1個胺基酸殘基的抗體等。 又，作為本發明之抗體，更具體而言，可列舉選自下述(a)～(l)中之任一種抗體。 (a)VH之CDR1～3之胺基酸序列分別為包含序列編號69、70及71所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號72、73及74所記載之胺基酸序列之胺基酸序列的抗體。 (b)VH之CDR1～3之胺基酸序列分別為包含序列編號75、76及77所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號78、79及80所記載之胺基酸序列之胺基酸序列的抗體。 (c)VH之CDR1～3之胺基酸序列分別為包含序列編號81、82及83所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號84、85及86所記載之胺基酸序列之胺基酸序列的抗體。 (d)VH之CDR1～3之胺基酸序列分別為包含序列編號87、88及89所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號90、91及92所記載之胺基酸序列之胺基酸序列的抗體。 (e)VH之CDR1～3之胺基酸序列分別為包含序列編號93、94及95所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號96、97及98所記載之胺基酸序列之胺基酸序列的抗體。 (f)VH之CDR1～3之胺基酸序列分別為包含序列編號99、100及101所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號102、103及104所記載之胺基酸序列之胺基酸序列的抗體。 (g)VH之CDR1～3之胺基酸序列分別為包含序列編號105、106及107所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號108、109及110所記載之胺基酸序列之胺基酸序列的抗體。 (h)VH之CDR1～3之胺基酸序列分別為包含序列編號111、112及113所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號114、115及116所記載之胺基酸序列之胺基酸序列的抗體。 (i)VH之CDR1～3之胺基酸序列分別為包含序列編號117、118及119所記載之胺基酸序列之胺基酸序列，且VL之CDR1～3之胺基酸序列分別為包含序列編號120、121及122所記載之胺基酸序列之胺基酸序列的抗體。 (j)與上述(a)～(i)記載之至少一種抗體競爭結合於人類CCR1的抗體。 (k)結合於包含上述(a)～(i)記載之任一種抗體所結合之表位之表位的抗體。 (l)結合於與上述(a)～(i)記載之任一種抗體所結合之表位相同之表位的抗體。 本發明之抗體包括具有如下抗體之VH之CDR1～3及VL之CDR1～3之胺基酸序列的抗體，該抗體之VH之CDR1～3及VL之CDR1～3之胺基酸序列分別與上述(a)～(i)所記載之任一種抗體之VH之CDR1～3及VL之CDR1～3之胺基酸序列表現出90%以上之同源性。所謂90%以上之同源性，具體而言，可列舉91%、92%、93%、94%、95%、96%、97%、98%及99%之同源性等。 本發明中，作為上述(a)～(i)所記載之抗體之一態樣，分別可列舉：小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體。又，作為上述(a)～(i)所記載之抗體之一態樣，分別可列舉：抗人類CCR1嵌合抗體chKM5907抗體、chKM5908抗體、chKM5909抗體、chKM5911抗體、chKM5915抗體、chKM5916抗體、chKM5954抗體、chKM5955抗體及chKM5956抗體。作為上述(a)～(i)所記載之抗體之一態樣，另外亦可列舉具有上述(a)～(i)所記載之任一種抗體之VH之CDR1～3及VL之CDR1～3之胺基酸序列的人類化抗體及人類抗體等。 本發明之上述(j)之抗體係指於將上述(a)～(i)記載之抗體設為第1抗體時，抑制該第1抗體與人類CCR1進行結合之第2抗體。本發明之上述(k)之抗體係指於將上述(a)～(i)記載之抗體設為第1抗體、及將第1抗體所結合之表位設為第1表位之情形時，結合於包含該第1表位之第2表位的第2抗體。又，本發明之上述(l)之抗體係指於將上述(a)～(i)記載之抗體設為第1抗體、及將第1抗體所結合之表位設為第1表位之情形時，結合於該第1表位之第2抗體。 又，作為本發明之抗體，具體而言，亦可列舉選自下述(a)～(i)中之任一種抗體。 (a)VH之胺基酸序列為包含序列編號51所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號52所記載之胺基酸序列之胺基酸序列的抗體。 (b)VH之胺基酸序列為包含序列編號53所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號54所記載之胺基酸序列之胺基酸序列的抗體。 (c)VH之胺基酸序列為包含序列編號55所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號56所記載之胺基酸序列之胺基酸序列的抗體。 (d)VH之胺基酸序列為包含序列編號57所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號58所記載之胺基酸序列之胺基酸序列的抗體。 (e)VH之胺基酸序列為包含序列編號59所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號60所記載之胺基酸序列之胺基酸序列的抗體。 (f)VH之胺基酸序列為包含序列編號61所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號62所記載之胺基酸序列之胺基酸序列的抗體。 (g)VH之胺基酸序列為包含序列編號63所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號64所記載之胺基酸序列之胺基酸序列的抗體。 (h)VH之胺基酸序列為包含序列編號65所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號66所記載之胺基酸序列之胺基酸序列的抗體。 (i)VH之胺基酸序列為包含序列編號67所記載之胺基酸序列之胺基酸序列，且VL之胺基酸序列為包含序列編號68所記載之胺基酸序列之胺基酸序列的抗體。 作為本發明之抗體，包括具有如下抗體之VH及VL之胺基酸序列的抗體，該抗體之VH及VL之胺基酸序列分別與上述(a)～(i)所記載之任一種抗體之VH及VL之胺基酸序列表現出90%以上之同源性。所謂90%以上之同源性，具體而言，可列舉：91%、92%、93%、94%、95%、96%、97%、98%及99%之同源性等。 本發明中，作為上述(a)～(i)所記載之抗體之一態樣，分別可列舉：小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體。 又，作為上述(a)～(i)所記載之抗體之一態樣，分別可列舉：抗人類CCR1嵌合抗體chKM5907抗體、chKM5908抗體、chKM5909抗體、chKM5911抗體、chKM5915抗體、chKM5916抗體、chKM5954抗體、chKM5955抗體及chKM5956抗體。 本發明中，作為人類CCR1，可列舉如下多肽，其包含序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列；或者如下多肽，其包含於序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列中1個以上之胺基酸缺失、經置換或附加的胺基酸序列，且具有人類CCR1之功能；或者如下多肽等，其包含與序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列具有60%以上、較佳為80%以上、更佳為90%以上、最佳為95%以上之同源性的胺基酸序列，且具有人類CCR1之功能。 具有於序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286所示之胺基酸序列中1個以上之胺基酸缺失、經置換或附加的胺基酸序列之多肽可藉由使用定點突變導入法[Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)、Nucleic acids Research, 10, 6487 (1982)、Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)、Gene, 34, 315 (1985)、Nucleic Acids Research, 13, 4431 (1985)、Proc. Natl. Acad. Sci. USA, 82, 488 (1985)]等，例如對編碼包含序列編號2之胺基酸序列之多肽的DNA導入定點突變而獲得。 缺失、經置換或附加之胺基酸之數量並無特別限定，較佳為1個～數十個，例如1～20個，更佳為1個～數個，例如1～5個胺基酸。 作為編碼人類CCR1之基因，可列舉序列編號1所記載之鹼基序列、及NCBI登錄號NM_001295之鹼基序列。本發明之編碼人類CCR1之基因亦包括：包含編碼如下多肽之DNA之基因，該多肽包含於序列編號1所記載之鹼基序列或NM_001295之鹼基序列中1個以上之鹼基缺失、經置換或附加的鹼基序列，且具有人類CCR1之功能；或者包含編碼如下多肽之DNA之基因，該多肽包含與序列編號1所記載之鹼基序列或NM_001295之鹼基序列至少具有60%以上之同源性的鹼基序列、較佳為具有80%以上之同源性的鹼基序列、更佳為具有95%以上之同源性的鹼基序列，且具有人類CCR1之功能；或者編碼如下多肽之基因等，該多肽包含與含有序列編號1所記載之鹼基序列或NM_001295之鹼基序列之DNA於嚴格條件下雜交的DNA，且具有人類CCR1之功能。 於嚴格條件下雜交之DNA係指使用包含序列編號1所記載之鹼基序列或NM_001295之鹼基序列的DNA作為探針，藉由菌落雜交法、噬菌斑雜交法、南方墨點雜交法或DNA微陣列法等而獲得之能夠進行雜交之DNA。 具體而言，可列舉能夠藉由如下方式鑑定之DNA，即，使用固定有源自經雜交之菌落或噬菌斑之DNA、或具有該序列之PCR產物或寡DNA的過濾器或載玻片，於0.7～1.0 mol/L之氯化鈉存在下，於65℃下進行雜交法[Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)、DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University, (1995)]後，使用0.1～2倍濃度之SSC溶液(1倍濃度之SSC溶液之組成包括150 mmol/L氯化鈉、15 mmol/L檸檬酸鈉)，於65℃條件下清洗過濾器或載玻片，藉此進行鑑定。 作為能夠進行雜交之DNA，可列舉：與序列編號1所記載之鹼基序列或NM_001295之鹼基序列至少具有60%以上之同源性的DNA、較佳為具有80%以上之同源性的DNA、更佳為具有95%以上之同源性的DNA。 編碼真核生物之蛋白質的基因之鹼基序列中常可見基因之多型。本發明之編碼人類CCR1之基因亦包括於本發明中所使用之基因中因上述多型而使鹼基序列發生小規模變異所得的基因。 關於本發明中之同源性之數值，除了特別明示之情況以外，可為使用業者公知之同源性檢索程式所算出之數值，針對鹼基序列，可列舉於BLAST[J. Mol. Biol., 215, 403 (1990)]中使用預設之參數所算出之數值等；針對胺基酸序列，可列舉於BLAST2[Nucleic Acids Res.,25, 3389 (1997)、Genome Res., 7, 649 (1997)、http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.htmL]中使用預設之參數所算出之數值等。 作為預設之參數，G(Cost to open gap)為鹼基序列之情形時係5，胺基酸序列之情形時係11；-E(Cost to extend gap)為鹼基序列之情形時係2，胺基酸序列之情形時係1；-q(Penalty for nucleotide mismatch)為-3、-r(reward for nucleotide match)為1、-e(expect value)為10、‑W(wordsize)為鹼基序列之情形時係11殘基，胺基酸序列之情形時係3殘基；-y[Dropoff(X)for blast extensions in bits]為blastn之情形時係20，blastn以外之程式之情形時係7；-X(X dropoff value for gapped alignment in bits)為15及-Z(final X dropoff value for gapped alignment in bits)為blastn之情形時係50，blastn以外之程式之情形時係25(http://www.ncbi.nlm.nih.gov/blast/htmL/blastcgihelp.htmL)。 包含序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列之部分序列的多肽可藉由業者公知之方法而製作。具體而言可藉由如下方式製作，即，使編碼序列編號2之胺基酸序列之DNA之一部分缺失，培養其中導入有包含該部分缺失DNA之表現載體的轉形體。又，可藉由與上述相同之方法而獲得如下多肽，該多肽具有於序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列中1以上之胺基酸缺失、經置換或附加的胺基酸序列。進而，包含序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列的多肽、或者具有於序列編號2所記載之胺基酸序列或NCBI登錄號NP_001286之胺基酸序列中1以上之胺基酸缺失、經置換或附加的胺基酸序列的多肽亦可藉由茀基甲氧基羰基(Fmoc)法、第三丁氧基羰基(tBoc)法等化學合成法製造。 作為本發明之抗體，亦包括多株抗體、單株抗體及寡株抗體中之任意之抗體。所謂多株抗體係指多純系之抗體產生細胞所分泌之抗體分子之群落。單株抗體係指單純系之抗體產生細胞所分泌之抗體，其僅識別一種表位(亦稱為epitope)，且構成單株抗體之胺基酸序列(一次序列)均勻。所謂寡株抗體係指將複數個不同之單株抗體混合而成之抗體分子之群落。 作為本發明中之單株抗體，可列舉：藉由融合瘤而產生之抗體、或由利用包含抗體基因之表現載體進行轉形而獲得之轉形體所產生之基因重組抗體。 所謂表位，可列舉：被單株抗體識別並結合之單一之胺基酸序列、包含胺基酸序列之立體結構、藉由轉譯後修飾進行修飾所得之胺基酸序列及包含該胺基酸序列之立體結構等。 作為藉由轉譯後修飾進行修飾所得之胺基酸序列，可列舉結合有O結合型糖鏈、N結合型糖鏈及硫酸基等之胺基酸序列，該O結合型糖鏈係糖鏈結合於具有OH取代基之Tyr及Ser者，該N結合型糖鏈係糖鏈結合於具有NH2
取代基之Gln及Asn者，該硫酸基係硫酸分子結合於具有OH取代基之Tyr者。 關於本發明之抗體於人類CCR1之細胞外區域之結合，可藉由使用ELISA、流式細胞儀及表面電漿子共振法等對本發明之抗體與人類CCR1表現細胞之結合性進行測定而確認。又，亦可組合公知之免疫學檢測法[Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996)、Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)，單選殖抗體實驗指南，Kodansha Scientific(1987)]等進行確認。 本發明之抗體所結合之人類CCR1之胺基酸殘基或表位可藉由使用使人類CCR1之一部分區域缺失所得之缺損體、與源自其他蛋白質之區域進行置換所得之變異體及人類CCR1之部分肽片段等，進行抗體之結合實驗而決定。又，亦可使用上述缺損體或變異體之表現細胞來進行抗體之結合實驗。 又，本發明之抗體所結合之人類CCR1之胺基酸殘基或表位亦可藉由如下方式決定，即，對經蛋白質分解酶消化之人類CCR1之肽片段添加本發明之抗體，使用已知之質譜分析法而進行表位映射。 關於本發明之抗體對由人類CCL15引起之人類CCR1之活化的抑制，例如可將人類CCR1表現細胞內之CCR1依賴性訊號傳遞、PLC之活化、細胞內之鈣離子濃度上升、NF-κB活化、或人類CCR1表現細胞之遷移作為指標進行確認。 細胞之遷移可使用以下記載之趨化性分析而測定。例如於趨化性分析腔室之上部添加人類CCR1表現細胞，於該腔室之下部分別添加1)培養基或DPBS等陰性對照、2)人類CCL15、及3)人類CCL15與本發明之抗體，培養一定小時後，利用適宜方法測定存在於該腔室下部之人類CCR1表現細胞數。關於所得之結果，若於添加有人類CCL15時之細胞數較添加有培養基時之細胞數而有所增加的條件下，添加有人類CCL15與本發明之抗體時之細胞數較添加有人類CCL15時之細胞數而有所減少，則可判定本發明之抗體抑制由人類CCL15引起之人類CCR1之活化。 又，本發明之抗體對由人類CCL15引起之人類CCR1之活化的抑制可將人類CCR1表現細胞內之鈣離子濃度之變化作為指標而確認。細胞內之鈣濃度之變化可藉由公知方法測定，例如可使用細胞內Ca測定套組(Wako公司製造)等，依據隨附之操作說明進行測定。 作為確認方法，例如可依據上述方法測定對人類CCR1表現細胞分別添加1)培養基或DPBS等陰性對照、2)人類CCL15、及3)人類CCL15與本發明之抗體時之細胞內鈣濃度之變化。若於添加有人類CCL15時之細胞內鈣離子濃度較添加有培養基時之細胞內鈣離子濃度而有所增加的條件下，添加有人類CCL15與本發明之抗體時之細胞內鈣離子濃度較添加有人類CCL15時之細胞內鈣離子濃度而有所減少，則可判定本發明之抗體抑制由人類CCL15引起之人類CCR1之活化。 抗體分子亦稱為免疫球蛋白(以下記為Ig)，人類抗體根據分子結構之不同，分為IgA1、IgA2、IgD、IgE、IgG1、IgG2、IgG3、IgG4及IgM之同型。亦將胺基酸序列之同源性相對較高之IgG1、IgG2、IgG3及IgG4統稱為IgG。 抗體分子係由稱為重鏈(Heavy chain，以下記為H鏈)及輕鏈(Light chain，以下記為L鏈)之多肽構成。又，H鏈係自N末端側起由VH、H鏈恆定區(亦記為CH)之各區構成，L鏈係自N末端側起由VL、L鏈恆定區(亦記為CL)之各區構成。CH對應於各亞型，分別已知α、δ、ε、γ及μ鏈。CH進而自N末端側起由CH1區、鉸鏈區、CH2區、CH3區之各區構成。所謂區(domain)係指構成抗體分子之各多肽的功能性結構單元。又，將CH2區與CH3區統稱為Fc區域或簡稱為Fc。CL已知Cλ
鏈及Cκ
鏈。 本發明中之CH1區、鉸鏈區、CH2區、CH3區及Fc區域可依據EU索引[Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]，根據自N末端起之胺基酸殘基之編號而加以特定。具體而言，將CH1特定為EU索引第118～215號之胺基酸序列、鉸鏈特定為EU索引第216～230號之胺基酸序列、CH2特定為EU索引第231～340號之胺基酸序列、CH3特定為EU索引第341～447號之胺基酸序列。 作為本發明之抗體，尤其亦包括藉由基因工程所製作之重組小鼠抗體、重組大鼠抗體、重組兔抗體、人類型嵌合抗體(以下亦簡稱為嵌合抗體)、人類化抗體(亦稱為人類型互補決定區CDR移植抗體)及人類抗體等基因重組抗體。 所謂嵌合抗體意指包含人類以外之動物(非人類動物)之抗體之VH及VL、與人類抗體之CH及CL的抗體。關於非人類動物，只要能夠製作融合瘤，可使用小鼠、大鼠、倉鼠、兔等任意動物。 所謂融合瘤係指使對非人類動物免疫抗原所取得之B細胞、與源自小鼠等之骨髓瘤細胞進行細胞融合而獲得的產生具有所需抗原特異性之單株抗體的細胞。因此，構成由融合瘤產生之抗體之可變區包含非人類動物抗體之胺基酸序列。 人類型嵌合抗體可藉由如下方式製造，即，自生產單株抗體之源自非人類動物細胞之融合瘤取得編碼該單株抗體之VH及VL之cDNA，分別插入至具有編碼人類抗體之CH及CL之DNA的動物細胞用表現載體而構建人類型嵌合抗體表現載體，導入至動物細胞中，藉此使之表現而製造。 所謂人類化抗體係指將非人類動物抗體之VH及VL之CDR之胺基酸序列移植入人類抗體之VH及VL之對應之CDR所得之抗體。VH及VL之CDR以外之區域稱為構架區(以下記為FR)。 人類化抗體可藉由如下方式製造，即，構建編碼如下VH之胺基酸序列之cDNA與編碼如下VL之胺基酸序列之cDNA，分別插入至具有編碼人類抗體之CH及CL之DNA的動物細胞用表現載體而構建人類化抗體表現載體，導入至動物細胞，藉此使之表現而製造，上述VH之胺基酸序列包含非人類動物抗體之VH之CDR之胺基酸序列與任意之人類抗體之VH之FR之胺基酸序列，上述VL之胺基酸序列包含非人類動物抗體之VL之CDR之胺基酸序列與任意之人類抗體之VL之FR之胺基酸序列。 人類抗體原本係指天然存在於人體內之抗體，但亦包括由近來隨著基因工程、細胞工程、發生工程技術之進步而製作之人類抗體噬菌體基因庫及產生人類抗體之基因轉殖動物所獲得的抗體等。 人類抗體可藉由使保持人類免疫球蛋白基因之小鼠(Tomizuka K. et. al., Proc Natl Acad Sci U S A. 97, 722-7, 2000)對所需抗原產生免疫而取得。又，藉由使用自源自人類之B細胞擴增抗體基因而得之噬菌體呈現(phage display)基因庫，而選擇具有所需結合活性之人類抗體，藉此不經免疫而可取得人類抗體(Winter G. et. al., Annu Rev Immunol.12:433-55. 1994)。進而，可使用EB病毒而使人類B細胞不死化，藉此製作生產具有所需結合活性之人類抗體之細胞而取得人類抗體(Rosen A. et. al., Nature 267, 52-54.1977)。 關於存在於人體內之抗體，例如藉由使自人類末梢血液單離出之淋巴球感染EB病毒等而使之不死化後，進行選殖，藉此可獲得產生該抗體之淋巴球，可自培養有該淋巴球之培養物中精製該抗體。 人類抗體噬菌體基因庫係藉由將由人類B細胞製備之抗體基因插入至噬菌體基因而實現於表面表現Fab、scFv等抗體片段之噬菌體之基因庫。可自該基因庫中，以對固定有抗原之受質之結合活性作為指標，而回收表現具有所需抗原結合活性之抗體片段之噬菌體。亦可進而藉由基因工程方法而將該抗體片段轉換成包含兩條完整之H鏈及兩條完整之L鏈之人類抗體分子。 產生人類抗體之基因轉殖動物係指將人類抗體基因組入至宿主動物之染色體內之動物。具體而言，對小鼠ES細胞導入人類抗體基因，將該ES細胞移植入其他小鼠之初期胚胎中後使之生長，藉此可製作產生人類抗體之基因轉殖動物。關於由產生人類抗體之基因轉殖動物製作人類抗體之方法，可藉由使用通常之人類以外之哺乳動物進行之融合瘤製作方法而取得產生人類抗體之融合瘤，加以培養，藉此使培養物中產生人類抗體並不斷蓄積。 作為本發明之抗體之VH及VL之胺基酸序列，可為人類抗體之VH及VL之胺基酸序列、非人類動物抗體之VH及VL之胺基酸序列、或者將非人類動物抗體之CDR移植入任意之人類抗體之構架區而得之人類化抗體之VH及VL之胺基酸序列中的任意者。 作為本發明之抗體中之CL之胺基酸序列，可為人類抗體之胺基酸序列或非人類動物抗體之胺基酸序列的任意者，較佳為人類抗體之胺基酸序列之Cκ
或Cλ
。 作為本發明之抗體之CH，只要屬於免疫球蛋白，則可為任意，較佳為屬於IgG類之亞型，可使用γ1(IgG1)、γ2(IgG2)、γ3(IgG3)及γ4(IgG4)中之任意者。 作為本發明之抗體，本發明中亦包括由Fc與抗體片段結合而成之Fc融合蛋白質、由Fc與天然存在之配體或受體結合而成之Fc融合蛋白質(亦稱為免疫黏附素)、使複數個Fc區域融合而成之Fc融合蛋白質等。又，為了使抗體保持穩定及為了控制血中半衰期，將胺基酸殘基改形之Fc區域等亦可用於本發明之抗體。 本發明之抗體或該抗體片段亦包括包含經轉譯後修飾之某胺基酸殘基之抗體。作為轉譯後修飾，例如可列舉：H鏈之C末端處之離胺酸殘基之缺失[離胺酸・剪切(lysine clipping)]或多肽之N末端處之麩醯胺殘基向焦麩醯胺(pyroGlu)之轉化等[Beck et al, Analytical Chemistry, 85, 715-736(2013)]。 本發明中，所謂抗體片段係結合於人類CCR1之細胞外區域而抑制由人類CCL15引起之人類CCR1之活化的具有抗原結合活性者。作為本發明中之抗體片段，可列舉：Fab、Fab'、F(ab')2
、scFv、diabody、dsFv或包含複數個CDR之肽等。Fab係於IgG抗體經木瓜蛋白分解酶處理所得之片段中(H鏈之第224號胺基酸殘基處被切斷)，H鏈之N末端側約一半與L鏈整體以雙硫鍵(S-S鍵)結合而獲得之分子量約5萬之具有抗原結合活性之抗體片段。 F(ab')2
係於IgG經胃蛋白分解酶處理所得之片段中(H鏈之第234號胺基酸殘基處被切斷)，略大於由Fab經鉸鏈區之S-S鍵結合而成者之分子量約10萬之具有抗原結合活性之抗體片段。Fab'係將上述F(ab')2
之鉸鏈區之S-S鍵切斷而獲得之分子量約5萬之具有抗原結合活性之抗體片段。 scFv係使用適宜之肽連結子(P)將1條VH與1條VL進行連結而獲得之VH-P-VL或VL-P-VH多肽形式之具有抗原結合活性之抗體片段，上述肽連結子(P)係包含4個Gly及1個Ser殘基之連結子(G4S)以任意個數相連而成之連結肽等。 Diabody係由抗原結合特異性相同或不同之scFv形成二聚物而獲得之對相同抗原具有二價抗原結合活性或對不同抗原具有特異性抗原結合活性之抗體片段。 dsFv係指使分別將VH及VL中之1個胺基酸殘基置換為半胱胺酸殘基所得之多肽經由該半胱胺酸殘基間之S-S鍵而結合而獲得者。 包含CDR之肽係包含VH或VL之CDR之至少一區域以上而構成。包含複數個CDR之肽可使CDR彼此直接結合或經由適宜之肽連接子而結合。可構建編碼本發明之改形抗體之VH及VL之CDR的DNA，將該DNA插入至原核生物用表現載體或真核生物用表現載體，將該表現載體導入至原核生物或真核生物中，藉此使之表現而製造。又，包含CDR之肽亦可藉由Fmoc法或tBoc法等化學合成法製造。 本發明之單株抗體包括結合於本發明之人類CCR1之單株抗體、或者藉由化學方法或基因工程使該抗體片段與放射性同位素、低分子之藥劑、高分子之藥劑、蛋白質或抗體醫藥等結合而獲得之抗體之衍生物。 抗體之衍生物可藉由利用化學方法[抗體工學入門，地人書館(1994)]使結合於本發明之人類CCR1之單株抗體或者該抗體片段之H鏈或L鏈之N末端側、C末端側、抗體分子中之適宜之取代基或側鏈或糖鏈等與放射性同位素、低分子之藥劑、高分子之藥劑、免疫活化劑、蛋白質、抗體醫藥或核酸醫藥等結合而製造。 又，亦可藉由如下基因工程方法而製造，即，使編碼結合於本發明之人類CCR1之單株抗體或該抗體片段的DNA、與編碼所欲結合之蛋白質或抗體醫藥的DNA進行連結並插入至表現載體，將該表現載體導入至適宜之宿主細胞中而使之表現。 作為放射性同位素，例如可列舉：111
In、131
I、125
I、90
Y、64
Cu、99
Tc、77
Lu或211
At等。放射性同位素可藉由氯胺T法等而直接結合於抗體。又，亦可使螯合有放射性同位素之物質結合於抗體。作為螯合劑，例如可列舉3-甲基二伸乙基三胺五乙酸1-異硫氰酸基苄酯(MX-DTPA)等。 作為低分子之藥劑，例如可列舉：烷基化劑、亞硝基脲劑、代謝拮抗劑、抗生素、植物鹼、拓樸異構酶抑制劑、激素療法劑、激素拮抗劑、芳香酶抑制劑、P糖蛋白抑制劑、鉑錯合物衍生物、M期抑制劑或激酶抑制劑等抗癌劑[臨床腫瘤學，癌與化學療法社(1996)]、氫化可體松或潑尼松等類固醇劑、阿司匹林或吲哚美辛等非類固醇劑、硫代蘋果酸金鈉或青黴胺等免疫調節劑、環磷醯胺或硫唑嘌呤等免疫抑制劑或順丁烯二酸氯苯那敏或氯馬斯汀(clemastine)之類的抗組織胺劑等抗炎症劑[炎症與抗炎症療法，醫齒藥出版股份有限公司(1982)]等。 作為抗癌劑，例如可列舉：胺磷汀(amifostine)(益護爾(Ethyol))、順鉑、達卡巴嗪(DTIC)、放線菌素(dactinomycin)、氮芥(Mechlorethamine/nitrogen mustard)、鏈脲黴素(Streptozocin)、環磷醯胺、異環磷醯胺、卡莫司汀(BCNU)、羅痰芥(CCNU)、多柔比星(阿德力黴素)、表柔比星、吉西他濱(健澤(Gemzar))、道諾黴素、普魯苄肼(Procarbazine)、絲裂黴素、阿糖胞苷、依託泊苷、甲胺喋呤、5-氟尿嘧啶、氟尿嘧啶、長春花鹼、長春新鹼、博萊黴素、道諾黴素(Daunomycin)、培洛黴素、雌莫司汀、紫杉醇(Taxo)、歐洲紫杉醇(Taxotere)、阿地白介素(aldesleukin)、天冬醯胺酶(asparaginase)、白消安、卡鉑、奧沙利鉑、奈達鉑(Nedaplatin)、克拉屈濱(cladribine)、喜樹鹼、10-羥基-7-乙基-喜樹鹼(SN38)、氟尿苷(Floxuridine)、氟達拉濱、羥基脲、依達比星、美司那(Mesna)、伊立替康(CPT-11)、拓樸替康(nogitecan)、米托蒽醌、拓朴替康(topotecan)、亮丙瑞林(Leuprolide)、甲地孕酮、美法侖、巰嘌呤、羥基脲(Hydroxycarbamide)、普卡黴素(plicamycin)、米托坦(mitotane)、培門冬酶(pegaspargase)、噴司他汀(Pentostatin)、哌泊溴烷(pipobroman)、他莫昔芬、戈舍瑞林、亮丙瑞林(leuprorelin)、氟他胺、替尼泊苷、睾丸內脂(Testolactone)、硫鳥嘌呤(thioguanine)、噻替派、尿嘧啶芥(uracil mustard)、長春瑞濱、氯芥苯丁酸(Chlorambucil)、氫化可體松、潑尼松龍、甲基潑尼松龍、長春地辛、尼莫司汀、司莫司汀(Semustine)、卡培他濱、雷替曲塞(Tomudex)、阿紮胞苷、UFT、oxaloplatin、吉米沙星(易瑞沙(Iressa))、伊馬替尼(STI571)、埃羅替尼、FMS樣酪胺酸激酶3(Flt3，FMS-like tyrosine kinase 3)抑制劑、血管內皮生長因子受體(VEGFR，vascular endothelial growth facotr receptor)抑制劑、纖維母細胞生長因子受體(FGFR，fibroblast growth factor receptor)抑制劑、易瑞沙或特羅凱(Tarceva)等表皮生長因子受體(EGFR，epidermal growth factor receptor)抑制劑、根赤殼菌素(Radicicol)、17-烯丙基胺基-17-去甲氧基膠達納黴素、雷帕黴素、安吖啶(Amsacrine)、全反式視黃酸、沙立度胺(Thalidomide)、來那度胺、阿那曲唑、法倔唑、來曲唑、依西美坦、硫代蘋果酸金鈉、D-青黴胺、布西拉明(Bucillamine)、硫唑嘌呤、咪唑立賓、環孢靈、雷帕黴素、氫化可體松、貝瑟羅汀(泰格雷丁(Targretin))、他莫昔芬、地塞米松、助孕素類、雌激素類、阿那曲唑(瑞寧得(Arimidex))、柳培林(leuplin)、阿司匹林、吲哚美辛、塞來考昔、青黴胺、硫代蘋果酸金鈉、順丁烯二酸氯苯那敏、氯苯那敏、氯馬斯汀、維生素A酸、貝瑟羅汀、砷、硼替佐米、別嘌呤醇、卡奇黴素(Calicheamicin)、替伊莫單抗(Ibritumomab tiuxetan)、泰格雷丁(Targretin)、ozogamine、克拉黴素(Clarithromycin)、甲醯四氫葉酸(Leucovorin)、酮康唑(Ketoconazole)、胺基導眠能(Aminoglutethimide)、蘇拉明(Suramin)或類美登素(maytansinoid)或其衍生物等。 作為使低分子之藥劑與抗體結合之方法，例如可列舉經由戊二醛而使藥劑與抗體之胺基間結合之方法、或經由水溶性碳二醯亞胺而使藥劑之胺基與抗體之羧基結合之方法等。 作為高分子之藥劑，例如可列舉：聚乙二醇(以下記為PEG)、白蛋白、葡聚糖、聚氧乙烯、苯乙烯順丁烯二酸共聚物、聚乙烯吡咯啶酮、吡喃共聚物、或羥丙基甲基丙烯醯胺等。藉由使該等高分子化合物與抗體或該抗體片段結合，而可期待(1)對化學上、物理上或生物上各種因子之穩定性提高、(2)血中半衰期顯著延長、或(3)免疫原性消失或抑制抗體產生等效果[生物複合醫藥品，廣川書店(1993)]。 例如作為使PEG與抗體結合之方法，可列舉與PEG化修飾試劑反應之方法等[生物複合醫藥品，廣川書店(1993)]。作為PEG化修飾試劑，可列舉：對離胺酸之ε-胺基之修飾劑(日本專利特開昭61-178926號公報)、對天冬胺酸及麩醯胺酸之羧基之修飾劑(日本專利特開昭56-23587號公報)、或對精胺酸之胍基之修飾劑(日本特開平2-117920號公報)等。 作為免疫活化劑，可為已知之作為免疫佐劑之天然物，關於具體例，可列舉作為免疫提高藥劑之β(1→3)葡聚糖(例如香菇多糖或西佐糖(Sizofiran))或α半乳糖苷基神經醯胺(KRN7000)等。 作為蛋白質，例如可列舉使NK細胞、巨噬細胞或嗜中性球等免疫活性(適格)細胞活化之細胞激素或生長因子或毒素蛋白質等。 作為細胞激素或生長因子，例如可列舉：干擾素(以下記為IFN)-α、IFN-β、IFN-γ、介白素(以下記為IL)-2、IL-12、IL-15、IL-18、IL-21、IL-23、粒細胞菌落刺激因子(G-CSF)、粒細胞/巨噬細胞菌落刺激因子(GM-CSF)或巨噬細胞菌落刺激因子(M-CSF)等。作為毒素蛋白質，例如可列舉：蓖麻毒蛋白(Ricin)、白喉毒素或ONTAK等，亦包括為了調節毒性而對蛋白質導入變異而獲得之蛋白毒素。 作為抗體醫藥，例如可列舉對應於如下抗原之抗體，即，因與抗體結合而誘導細胞凋亡之抗原、腫瘤之病態形成之相關抗原、調節免疫功能之抗原或病變部位之血管新生之相關抗原。 作為因與抗體結合而誘導細胞凋亡之抗原，例如可列舉：白細胞分化抗原(cluster of differentiation，以下記載為CD)19、CD20、CD21、CD22、CD23、CD24、CD37、CD53、CD72、CD73、CD74、CDw75、CDw76、CD77、CDw78、CD79a、CD79b、CD80(B7.1)、CD81、CD82、CD83、CDw84、CD85、CD86(B7.2)、人類白細胞抗原II類(human leukocyte antigen(HLA)-Class II)或表皮生長因子受體(EGFR，Epidermal Growth Factor Receptor)等。 作為腫瘤之病態形成之相關抗原或調節免疫功能之抗體之抗原，例如可列舉：CD4、CD40、CD40配體、B7家族分子(例如CD80、CD86、CD274、B7-DC、B7-H2、B7-H3或B7-H4)、B7家族分子之配體(例如CD28、CTLA-4、ICOS、PD-1或BTLA)、OX-40、OX-40配體、CD137、腫瘤壞死因子(TNF，tumor necrosis factor)受體家族分子(例如DR4、DR5、TNFR1或TNFR2)、TNF相關之凋亡誘導配體(TRAIL，TNF-related apoptosis-inducing ligand receptor)家族分子、TRAIL家族分子之受體家族(例如TRAIL-R1、TRAIL-R2、TRAIL-R3或TRAIL-R4)、核因子κB受體活化因子(RANK，receptor activator of nuclear factor kappa B ligand)、RANK配體、CD25、葉酸受體、細胞激素[例如IL-1α、IL-1β、IL-4、IL-5、IL-6、IL-10、IL-13、轉型生長因子(TGF，transforming growth factor)β或TNFα等]或該等細胞激素之受體、或趨化素(例如SLC、ELC、I-309、TARC、MDC或CTACK等)或該等趨化素之受體。 作為抑制病變部位血管新生之抗體之抗原，例如可列舉：血管內皮生長因子(VEGF，vascular endothelial growth factor)、血管生成素(angiopoietin)、纖維母細胞生長因子(FGF，fibroblast growth factor)、EGF、肝細胞生長因子(HGF，hepatocyte growth factor)、血小板衍生生長因子(PDGF，platelet-derived growth factor)、胰島素樣生長因子(IGF，insulin-like growth factor)、紅血球生成素(EPO，erythropoietin)、TGFβ、IL-8、蝶素(ephrin)或SDF-1或該等之受體等。 關於與蛋白質或抗體醫藥之融合抗體，可使編碼單株抗體或抗體片段之cDNA與編碼蛋白質或抗體醫藥所含之抗體之cDNA連結，構建編碼融合抗體之DNA，將該DNA插入至原核生物或真核生物用表現載體，將該表現載體導入至原核生物或真核生物中，藉此使之表現而製造融合抗體。 作為核酸醫藥，例如可列舉包含藉由控制基因之功能而作用於生物體之小干擾核糖核酸(siRNA，small interference ribonucleic acid)或微小核糖核酸(microRNA)等核酸的醫藥品。例如可想到與抑制Th17細胞之主轉錄因子RORγt之核酸醫藥的複合物。 於將本發明之抗體之衍生物用於人類CCR1之檢測及測定以及人類CCR1相關疾病之診斷的情形時，作為結合於該抗體之藥劑，可列舉通常之免疫學檢測或測定法中所使用之標記物。作為標記物，例如可列舉：鹼性磷酸酶、過氧化酶或螢光素酶等酶，吖啶鎓酯或咯吩等發光物質，或者螢光異硫氰酸鹽(FITC)或異硫氰酸四甲基羅丹明(RITC)等螢光物質等。 又，本發明包括含有結合於人類CCR1之單株抗體或該抗體片段作為有效成分之組合物。 又，本發明係關於一種人類CCR1相關疾病之治療藥，其含有結合於人類CCR1之單株抗體或該抗體片段作為有效成分。又，本發明係關於一種人類CCR1相關疾病之治療方法，其包括投予結合於人類CCR1之單株抗體或該抗體片段。 作為人類CCR1相關疾病，可為與人類CCR1或人類CCR1之配體有關之任意疾病，例如可列舉：癌症、自體免疫疾病及炎症性疾病。作為癌症，例如可列舉：彌漫性大細胞型B細胞性淋巴瘤、濾胞性淋巴瘤、B細胞淋巴瘤、T細胞淋巴瘤、漿細胞性骨髓瘤、急性骨髄性白血病、霍奇金淋巴瘤、慢性淋巴性白血病、毛細胞白血病、套細胞淋巴瘤、濾胞邊緣帶淋巴瘤、小淋巴球性淋巴瘤、多發性骨髓瘤、肝細胞癌、結腸直腸癌、非小細胞肺癌、口腔扁平上皮癌、卵巢癌、前列腺癌、乳癌、神經膠質瘤或骨肉瘤等。作為自體免疫疾病或炎症性疾病，例如可列舉：類風濕性關節炎、多發性硬化症、慢性阻塞性肺病、全身性紅斑狼瘡、狼瘡性腎炎、哮喘、異位性皮膚炎、炎症性大腸炎、克隆氏病或貝西氏病等。 含有本發明之抗體或該抗體片段之治療劑可為僅包含作為有效成分之該抗體或該抗體片段者，但較佳為以與通常於藥理學上容許之1種以上之載體一併混合後藉由製劑學技術領域中公知之任意方法所製造之醫藥製劑的形式提供。 投予路徑較佳成為採用治療時最具效果者，可列舉：經口投予、或口腔內、氣管內、直腸內、皮下、肌內或靜脈內等非經口投予，較佳列舉靜脈內投予。作為投予形態，例如可列舉：噴霧劑、膠囊劑、錠劑、散劑、顆粒劑、糖漿劑、乳劑、栓劑、注射劑、軟膏或貼劑等。 投予量或投予次數根據目標治療效果、投予方法、治療時間、年齡及體重等而不同，通常情況下成人每日10 μg/kg～10 mg/kg。 本發明係關於一種含有結合於人類CCR1之單株抗體或該抗體片段的CCR1之檢測或測定用試劑、或使用結合於人類CCR1之單株抗體或該抗體片段的CCR1之檢測或測定方法。本發明中，作為檢測或測定人類CCR1之方法，可列舉任意公知之方法。例如可列舉免疫學檢測或測定方法等。 所謂免疫學檢測或測定方法係使用經標記之抗原或抗體而檢測或測定抗體量或抗原量之方法。作為免疫學檢測或測定方法，例如可列舉：放射性物質標記免疫抗體法(RIA)、酶免疫測定法(EIA或ELISA)、螢光免疫測定法(FIA)、發光免疫測定法(luminescent immunoassay)、西方墨點法或物理化學方法等。 本發明係關於一種CCR1相關疾病之診斷藥，其包含結合於人類CCR1之單株抗體或該抗體片段，或關於一種CCR1相關疾病之診斷方法，其包括使用結合於人類CCR1之單株抗體或該抗體片段而檢測或測定CCR1。藉由使用本發明之單株抗體或該抗體片段，依據上述方法而檢測或測定人類CCR1表現細胞，可診斷與人類CCR1相關之疾病。 本發明中，作為成為檢測或測定人類CCR1之對象的生物體試樣，只要為有可能包含人類CCR1或人類CCR1表現細胞者，則並無特別限定，例如組織、細胞、血液、血漿、血清、胰臟液、尿、糞便、組織液或培養液等。 含有本發明之單株抗體或該抗體片段之診斷藥根據目標之診斷法而可包含用以進行抗原抗體反應之試劑、該反應之檢測用試劑。作為用以進行抗原抗體反應之試劑，可列舉：緩衝劑、鹽等。作為檢測用試劑，可列舉：識別該單株抗體或該抗體片段之經標記之二次抗體、或與標記對應之受質等通常之免疫學檢測或測定法所使用之試劑。 又，本發明係關於一種抗人類CCR1單株抗體或該抗體片段之用途，其用於製造CCR1相關疾病之治療藥或診斷藥。 以下，對本發明之抗體之製造方法、疾病之治療方法及疾病之診斷方法進行具體說明。 1.抗體之製造方法 (1)抗原之製備 成為抗原之人類CCR1或人類CCR1表現細胞可藉由將包含編碼人類CCR1全長或部分長度之cDNA的表現載體導入至大腸桿菌、酵母、昆蟲細胞或動物細胞等中而獲得。又，人類CCR1亦可藉由自大量表現人類CCR1之各種人類細胞株、人類細胞及人類組織等精製人類CCR1而獲得。又，亦可將該等人類細胞株、人類細胞及人類組織等直接用作抗原。進而，亦可藉由Fmoc法或tBoc法等化學合成法製備具有人類CCR1之部分序列之合成肽而用於抗原。對於人類CCR1或具有人類CCR1之部分序列之合成肽，亦可於C末端或N末端附加FLAG或His等公知之標籤。 本發明中使用之人類CCR1可採用Molecular Cloning,A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)或Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997)等中所記載之方法等，例如藉由以下之方法，使編碼該人類CCR1之DNA於宿主細胞中進行表現而製造。 首先，藉由將包含編碼人類CCR1之部分的全長cDNA插入至適當之表現載體之啟動子之下游，而製作重組載體。亦可使用基於全長cDNA而製備之包含編碼多肽之部分的適當長度之DNA片段代替上述全長cDNA。其次，將所獲得之該重組載體導入至適合該表現載體之宿主細胞中，藉此可獲得生產多肽之轉形體。 作為表現載體，只要為能夠於所使用之宿主細胞中進行自主複製或能夠組入至染色體中、且於可轉錄編碼多肽之DNA的位置含有適當之啟動子者，則均可使用。作為宿主細胞，只要為大腸桿菌等屬於埃希菌屬(Escherichia)等之微生物、酵母、昆蟲細胞或動物細胞等可表現目標基因者，則均可使用。 於使用大腸桿菌等原核生物作為宿主細胞之情形時，重組載體較佳為能夠於原核生物中進行自主複製且同時包含啟動子、核糖體結合序列、包含編碼人類CCR1之部分之DNA及轉錄終止序列的載體。又，對於該重組載體而言，轉錄終止序列並非必須，但較佳為於結構基因之正下游配置轉錄終止序列。進而，該重組載體亦可包含控制啟動子之基因。 作為該重組載體，較佳為使用將作為核糖體結合序列之Shine-Dalgarno序列(亦稱為SD序列)與起始密碼子之間調節為適當距離(例如6～18個鹼基)之質體。 又，關於編碼該人類CCR1之DNA之鹼基序列，可以成為最適於宿主內之表現之密碼子之方式置換鹼基，藉此可提高目標之人類CCR1之生產率。 作為表現載體，只要為能夠於所使用之宿主細胞中發揮功能者，則均可使用，例如可列舉：pBTrp2、pBTac1、pBTac2(以上為Roche Diagnostics公司製造)、pKK233-2(Pharmacia公司製造)、pSE280(Invitrogen公司製造)、pGEMEX-1(Promega公司製造)、pQE-8(Qiagen公司製造)、pKYP10(日本專利特開昭58-110600號公報)、pKYP200[Agricultural Biological Chemistry, 48, 669 (1984)]、pLSA1[Agric. Biol. Chem., 53, 277 (1989)]、pGEL1[Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)]、pBluescript II SK(-)(Stratagene公司製造)、pTrs30[由大腸桿菌JM109/pTrS30(FERM BP-5407)製備]、pTrs32[由大腸桿菌JM109/pTrS32(FERM BP-5408)製備]、pGHA2[由大腸桿菌IGHA2(FERM BP-400)製備，日本專利特開昭60-221091號公報]、pGKA2[由大腸桿菌IGKA2(FERM BP-6798)製備，日本專利特開昭60-221091號公報]、pTerm2(美國專利第4,686,191號說明書、美國專利第4,939,094號說明書、美國專利第160,735號說明書)、pSupex、pUB110、pTP5、pC194、pEG400[J. Bacteriol., 172, 2392 (1990)]、pGEX(Pharmacia公司製造)、pET系統(Novagen公司製造)或pME18SFL3等。 作為啟動子，只要為能夠於所使用之宿主細胞中發揮功能者，則可為任意者。例如可列舉：trp啟動子(Ptrp)、lac啟動子、PL啟動子、PR啟動子或T7啟動子等源自大腸桿菌或噬菌體等之啟動子。又，例如可列舉：使2個Ptrp串聯而成之串聯啟動子、tac啟動子、lacT7啟動子或let I啟動子等人工地進行設計改形之啟動子等。 作為宿主細胞，例如可列舉：大腸桿菌XL1-藍、大腸桿菌XL2-藍、大腸桿菌DH1、大腸桿菌MC1000、大腸桿菌KY3276、大腸桿菌W1485、大腸桿菌JM109、大腸桿菌HB101、大腸桿菌No.49、大腸桿菌W3110、大腸桿菌NY49或大腸桿菌DH5α等。 作為重組載體向宿主細胞之導入方法，只要為向所使用之宿主細胞中導入DNA之方法，則均可使用，例如可列舉使用鈣離子之方法[Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)、Gene, 17, 107 (1982)、Molecular & General Genetics, 168, 111 (1979)]。 於使用動物細胞作為宿主之情形時，作為表現載體，只要為能夠於動物細胞中發揮功能者，則均可使用，例如可列舉：pcDNAI、pCDM8(Funakoshi公司製造)、pAGE107[日本特開平3-22979號公報；Cytotechnology, 3, 133 (1990)]、pAS3-3(日本特開平2-227075號公報)、pCDM8[Nature, 329, 840 (1987)]、pcDNAI/Amp(Invitrogen公司製造)、pcDNA3.1(Invitrogen公司製造)、pREP4(Invitrogen公司製造)、pAGE103[J. Biochemistry, 101, 1307 (1987)]、pAGE210、pME18SFL3、pKANTEX93(國際公開第97/10354號)、N5KG1val(美國專利第6,001,358號說明書)、INPEP4(Biogen-IDEC公司製造)及轉位子載體(國際公開第2010/143698號)等。 作為啟動子，只要為能夠於動物細胞中發揮功能者，則均可使用，例如可列舉：巨細胞病毒(CMV)之即刻早期(IE，immediate early)基因之啟動子、SV40之初期啟動子、反轉錄病毒之啟動子、金屬硫蛋白啟動子、熱休克啟動子、SRα啟動子或莫洛尼(Moloney)小鼠白血病病毒之啟動子或促進子。又，亦可將人類CMV之IE基因之促進子與啟動子一併使用。 作為宿主細胞，例如可列舉：人類白血病細胞Namalwa細胞、猴細胞COS細胞、中國倉鼠卵巢細胞CHO細胞[Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60 , 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900)]；二氫葉酸還原酶基因(以下，記為dhfr)缺損之CHO細胞(CHO/DG44細胞)[Proc.Natl.Acad.Sci.USA,77,4216(1980)]、CHO-K1(ATCC CCL-61)、DUkXB11(ATCC CCL-9096)、Pro-5(ATCC CCL-1781)、CHO-S(Life Technologies、Cat#11619)、Pro-3、大鼠骨髓瘤細胞YB2/3HL.P2.G11.16Ag.20(或亦稱為YB2/0)、小鼠骨髓瘤細胞NSO、小鼠骨髓瘤細胞SP2/0-Ag14、敘利亞倉鼠細胞BHK或HBT5637(日本專利特開昭63-000299號公報)等。 作為重組載體向宿主細胞之導入方法，只要為向動物細胞導入DNA之方法，則均可使用，例如可列舉：電穿透法[Cytotechnology, 3, 133 (1990)]、磷酸鈣法(日本特開平2-227075號公報)或脂轉染法[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]等。 藉由如上方式獲得保有組入了編碼人類CCR1之DNA之重組載體的微生物或動物細胞等，將源自該微生物或動物細胞等之轉形體於培養基中加以培養，使培養液中生成該人類CCR1並不斷蓄積，自該培養液進行採集，藉此可製造人類CCR1。於培養基中培養該轉形體之方法可依據宿主之培養所使用之通常方法進行。 於源自真核生物之細胞中進行表現之情形時，可獲得附加有糖或糖鏈之人類CCR1。 於對利用使用誘導性啟動子之重組載體進行轉形之微生物加以培養時，視需要可於培養基中添加誘導體。例如於對利用使用lac啟動子之重組載體進行轉形之微生物加以培養之情形時，可於培養基中添加異丙基-β-D-硫代半乳糖苷等，於對利用使用trp啟動子之重組載體進行轉形之微生物加以培養之情形時，可於培養基中添加吲哚丙烯酸等。 作為培養以動物細胞為宿主而獲得之轉形體的培養基，例如可列舉：通常使用之RPMI1640培養基[The Journal of the American Medical Association, 199, 519 (1967)]、Eagle之MEM培養基[Science, 122, 501 (1952)]、杜爾貝科改良MEM培養基[Virology, 8, 396 (1959)]、199培養基[Proc. Soc. Exp. Biol. Med., 73, 1 (1950)]或Iscove改良杜爾貝科培養基(IMDM，Iscove's Modified Dulbecco's Medium)培養基或於該等培養基中添加有胎牛血清(FBS)等之培養基等。培養通常係於pH值6～8、30～40℃、5%CO2
之存在下等條件下進行1～7天。又，於培養過程中，視需要亦可對培養基添加康黴素或青黴素等抗生素。 作為編碼人類CCR1之基因之表現方法，例如除了直接表現以外，亦可列舉分泌生產或融合蛋白質表現等方法[Molecular Cloning, A Laboratory Manual,Second Edition,Cold Spring Harbor Laboratory Press (1989)]。 作為人類CCR1之生產方法，例如可列舉：於宿主細胞內生產之方法、分泌至宿主細胞外之方法、或於宿主細胞外膜上生產之方法，可藉由改變所使用之宿主細胞或欲生產之人類CCR1之結構而選擇適宜方法。 於宿主細胞內或宿主細胞外膜上生產人類CCR1之情形時，可藉由使用Paulson等人之方法[J. Biol. Chem., 264, 17619 (1989)]、Laura等人之方法[Proc. Natl. Acad. Sci., USA, 86, 8227 (1989)、Genes Develop., 4, 1288 (1990)]、日本特開平05-336963號公報或國際公開第94/23021號等所記載之方法，而使人類CCR1積極地分泌至宿主細胞外。又，亦可利用使用二氫葉酸還原酶基因等之基因擴增系(日本特開平2-227075號公報)而使人類CCR1之生產量提昇。 所獲得之人類CCR1例如可藉由以下方式進行單離、精製。於人類CCR1在細胞內以溶解狀態表現之情形時，培養結束後藉由離心分離而回收細胞，使之懸浮於水系緩衝液中，其後使用超音波破碎機、壓滲機(Fresh press)、Manton-Gaulin均質機或珠磨機等破碎細胞而獲得無細胞萃取液。藉由對該無細胞萃取液進行離心分離而獲得上清液，可單獨使用或組合使用通常之蛋白質之單離精製法，即溶劑萃取法、利用硫酸銨等之鹽析法、脫鹽法、利用有機溶劑之沈澱法、使用二乙基胺基乙基(DEAE)- 瓊脂糖凝膠(Sepharose)、DIAION HPA-75(三菱化學公司製造)等樹脂之陰離子交換層析法、使用S-瓊脂糖FF(Pharmacia公司製造)等樹脂之陽離子交換層析法、使用丁基瓊脂糖凝膠、苯基瓊脂糖凝膠等樹脂之疏水性層析法、使用分子篩之凝膠過濾法、親和層析法、層析聚焦法(chromatofocusing)、或等電點電泳等電泳法等方法，而由上述上清液獲得精製標本。 於人類CCR1在細胞內形成不溶體而表現之情形時，藉由與上述相同之方式回收細胞後加以破碎，並進行離心分離，藉此回收作為沈澱部分之該人類CCR1之不溶體。利用蛋白質改性劑使所回收之該人類CCR1之不溶體可溶化。藉由將該可溶化液稀釋或透析而使該人類CCR1恢復正常之立體結構後，藉由與上述相同之單離精製法，而可獲得多肽之精製標本。 於人類CCR1或其糖修飾體等衍生物被分泌至細胞外之情形時，可於培養上清液中回收該人類CCR1或其糖修飾體等衍生物。可藉由與上述同樣地利用離心分離等方法處理該培養物而獲得可溶性部分，藉由使用與上述相同之單離精製法而自該可溶性部分獲得精製標本。 又，本發明中使用之人類CCR1亦可藉由Fmoc法或tBoc法等化學合成法而製造。又，亦可利用Advanced Chemtec公司製造、Perkin Elmer公司製造、Pharmacia公司製造、Protein Technology Instrument公司製造、Synthecell-Vega公司製造、Per Septive公司製造或島津製作所公司製造等之肽合成機而進行化學合成。 (2)動物之免疫與融合用抗體產生細胞之製備 對於3～20週齡之小鼠、大鼠或倉鼠等動物，利用(1)中所獲得之抗原進行免疫，自該動物之脾臟、淋巴結、末梢血液中採集抗體產生細胞。又，亦可使用小鼠CCR1剔除小鼠作為被免疫動物。 免疫係藉由將抗原與例如弗氏完全佐劑、或氫氧化鋁凝膠及百日咳菌疫苗等適當之佐劑一併投予至動物之皮下、靜脈內或腹腔內而進行。於抗原為部分肽之情形時，製作與BSA(牛血清白蛋白)或KLH(Keyhole Limpet hemocyanin，鑰孔蟲戚血藍蛋白)等載體蛋白之複合物，並將其用作免疫原。 抗原之投予係於第1次投予後，以1～2週為間隔投予5～10次。各投予後第3～7天自眼底靜脈叢採血，使用酶免疫測定法[Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]等測定該血清之抗體效價。將血清對於免疫所使用之抗原顯示出充分之抗體效價之動物作為融合用抗體產生細胞之供給源。 於抗原之最終投予後第3～7天，自經免疫動物摘取脾臟等包含抗體產生細胞之組織，採集抗體產生細胞。於使用脾臟細胞之情形時，將脾臟切碎、離散後，進行離心分離，進而去除紅血球，而獲得融合用抗體產生細胞。 (3)骨髓瘤細胞之製備 作為骨髓瘤細胞，使用自小鼠獲得之株化細胞，例如使用8-氮鳥嘌呤耐性小鼠(源自BALB/c)骨髓瘤細胞株P3-X63Ag8-U1(P3-U1)[Current Topics in Microbiology and Immunology, 18, 1 (1978)]、P3-NS1/1-Ag41(NS-1)[European J. Immunology, 6, 511 (1976)]、SP2/0-Ag14(SP-2)[Nature, 276, 269 (1978)]、P3-X63-Ag8653(653)[J. Immunology, 123, 1548 (1979)]或P3-X63-Ag8(X63)[Nature, 256, 495 (1975)]等。 該骨髓瘤細胞於正常培養基[添加有麩醯胺、2-巰基乙醇、慶大黴素、FBS及8-氮鳥嘌呤之RPMI1640培養基]中進行繼代，於細胞融合之3～4天前使之繼代至正常培養基中，確保融合當天細胞數有2×107
個以上。 (4)細胞融合與單株抗體產生融合瘤之製備 將(2)中所獲得之融合用抗體產生細胞與(3)中所獲得之骨髓瘤細胞利用最低必需培養基(MEM，Minimum Essential Medium)培養基或PBS(磷酸氫二鈉1.83 g、磷酸二氫鉀0.21 g、食鹽7.65 g、蒸餾水1 L，pH值7.2)充分洗淨，以細胞數為融合用抗體產生細胞：骨髓瘤細胞＝5～10：1之方式進行混合，進行離心分離後，去除上清液。使沈澱之細胞群充分地離散後，於37℃下一面攪拌一面添加聚乙二醇-1000(PEG-1000)、MEM培養基及二甲基亞碸之混合液。進而，每隔1～2分鐘添加1～2 mL之MEM培養基，添加數次後，以總量成為50 mL之方式添加MEM培養基。進行離心分離後，去除上清液。緩慢地使沈澱之細胞群離散後，使融合用抗體產生細胞緩慢地懸浮於HAT培養基[添加有次黃嘌呤、胸苷及胺喋呤之正常培養基]中。將該懸浮液於5%CO2
培養箱中於37℃下培養7～14天。 培養後，抽取一部分培養上清液，藉由下述結合分析等融合瘤之選擇方法，選出與包含人類CCR1之抗原進行反應且不與不含人類CCR1之抗原進行反應之細胞群。其次，藉由極限稀釋法進行選殖，選出穩定地確認到較強抗體效價者作為單株抗體產生融合瘤。 (5)精製單株抗體之製備 對經姥鮫烷處理[於腹腔內投予2,6,10,14-四甲基十五烷(Pristane)0.5 mL，飼養2週]之8～10週齡小鼠或裸小鼠，於腹腔內注射(4)中所獲得之單株抗體產生融合瘤。經過10～21天，融合瘤發生腹水癌化。自該小鼠採集腹水，進行離心分離，去除固形物成分後，利用40～50%硫酸銨進行鹽析，藉由辛酸沈澱法、DEAE-瓊脂糖凝膠管柱、蛋白質A-管柱或凝膠過濾管柱進行精製，收集IgG或IgM部分作為精製單株抗體。 又，亦可將(4)中所獲得之單株抗體產生融合瘤於添加有10%FBS之RPMI1640培養基等中進行培養後，藉由離心分離而去除上清液，使之懸浮於融合瘤SFM培養基中，培養3～7天。對所獲得之細胞懸浮液進行離心分離，利用蛋白質A-管柱或蛋白質G-管柱自所獲得之上清液進行精製，收集IgG部分，而獲得精製單株抗體。再者，亦可對融合瘤SFM培養基添加5%Daigo's GF21。 抗體之亞型之確定係使用亞型鑑定套組(subtype typing kit)並藉由酶免疫測定法而進行。蛋白質量之定量係根據洛利(Lowry)法或280 nm下之吸光度而算出。 (6)單株抗體之選擇 如下所示，單株抗體之選擇係藉由使用流式細胞儀測定抗體對人類CCR1表現細胞之結合性等而進行。人類CCR1表現細胞可為於細胞表面上表現人類CCR1之任意細胞，例如可列舉：人類細胞、人類細胞株及(1)中所獲得之人類CCR1強制表現細胞株等。 將人類CCR1表現細胞分注至96孔板等培養盤後，分注作為第1抗體之血清、融合瘤之培養上清液或精製單株抗體等被試驗物質，使之反應。將反應後之細胞利用包含1～10%牛血清白蛋白(BSA，bovine serum albumin)之PBS(以下記為BSA-PBS)等充分洗淨後，分注作為第2抗體之經螢光試劑等標記之抗免疫球蛋白抗體而使之反應。利用BSA-PBS等充分洗淨後，使用流式細胞儀測定標記化抗體之螢光量，藉此選擇特異性地與人類CCR1表現細胞進行反應之單株抗體。 又，與本發明之抗體競爭結合於人類CCR1之抗體可藉由對上述使用流式細胞儀之測定系統添加被試驗抗體並使之反應而取得。即，篩選於添加有被試驗抗體時本發明之抗體與人類CCR1之結合受到抑制的抗體，藉此可取得與本發明之抗體競爭結合於人類CCR1之胺基酸序列或其立體結構之單株抗體。 又，結合於包含本發明之與人類CCR1結合之單株抗體所結合之表位在內之表位上之抗體可藉由如下方式取得，即，藉由公知方法鑑定利用上述篩選方法所取得之抗體之表位，製作包含所鑑定出之表位之合成肽、或模擬表位立體結構之合成肽等，進行免疫。 進而，結合於與本發明之與人類CCR1結合之單株抗體所結合之表位相同之表位上之抗體可藉由如下方式取得，即，鑑定利用上述篩選方法所取得之抗體之表位，製作所鑑定出之表位之部分合成肽、或模擬表位立體結構之合成肽等，進行免疫。 2.基因重組抗體之製作 作為基因重組抗體之製作例，以下揭示人類型嵌合抗體及人類化抗體之製作方法。基因重組之小鼠抗體、大鼠抗體及兔抗體等亦可藉由相同方法製作。 (1)基因重組抗體表現用載體之構建 基因重組抗體表現用載體係組入有編碼人類抗體之CH及CL之DNA的動物細胞用表現載體，可藉由將編碼人類抗體之CH及CL之DNA分別選殖至動物細胞用表現載體而構建。 人類抗體之C區域可使用任意之人類抗體之CH及CL。例如使用人類抗體之γ1亞型之CH及κ型之CL等。編碼人類抗體之CH及CL之DNA係使用cDNA，但亦可使用包含外顯子與內含子之染色體DNA。作為動物細胞用表現載體，只要為能夠組入並表達編碼人類抗體之C區域之基因者則均可使用。例如使用pAGE107[Cytotechnol., 3, 133 (1990)]、pAGE103[J. Biochem., 101, 1307 (1987)]、pHSG274[Gene, 27, 223 (1984)]、pKCR[Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)]、pSG1bd2-4[Cytotechnol., 4, 173 (1990)]或pSE1UK1Sed1-3[Cytotechnol., 13, 79 (1993)]等。作為動物細胞用表現載體中之啟動子與促進子，可列舉：SV40之初期啟動子[J. Biochem., 101, 1307 (1987)]、莫洛尼小鼠白血病病毒LTR[Biochem. Biophys. Res. Commun., 149, 960 (1987)]或免疫球蛋白H鏈之啟動子[Cell, 41, 479 (1985)]與促進子[Cell, 33, 717 (1983)]等。 就基因重組抗體表現載體之易構建性、向動物細胞之易導入性、動物細胞內之抗體H鏈及L鏈之表現量之均衡性等方面而言，基因重組抗體表現用載體可使用抗體H鏈及L鏈存在於同一載體上之類型(串聯型)之基因重組抗體表現用載體[J.Immunol. Methods， 167， 271(1994)，亦可使用抗體H鏈及L鏈存在於不同載體上之類型。作為串聯型之基因重組抗體表現用載體，使用pKANTEX93(國際公開第97/10354號)、pEE18[Hybridoma, 17, 559 (1998)]等。 (2)編碼源自人類以外之動物之抗體之V區域的cDNA之取得及胺基酸序列之解析 編碼非人類抗體之VH及VL之cDNA之取得及胺基酸序列之解析可藉由如下方式進行。 自產生非人類抗體之融合瘤細胞萃取mRNA而合成cDNA。將所合成之cDNA選殖至噬菌體或質體等載體而製作cDNA基因庫。使用編碼小鼠抗體之C區域部分或V區域部分之DNA作為探針，分別自該基因庫中單離具有編碼VH或VL之cDNA之重組噬菌體或重組質體。分別確定重組噬菌體或重組質體上之目標小鼠抗體之VH或VL之全鹼基序列，根據鹼基序列而分別推測VH或VL之全胺基酸序列。 製作可產生非人類抗體之融合瘤細胞的人類以外之動物係使用小鼠、大鼠、倉鼠或兔等，可使用能夠製作融合瘤細胞之任意動物。 由融合瘤細胞製備全RNA時使用硫氰酸胍-三氟乙酸銫法[Methods in Enzymol., 154, 3 (1987)]或RNA easy kit(Qiagen公司製造)等套組等。 由全RNA製備mRNA時使用寡(dT)固定化纖維素管柱法[Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]、或Oligo-dT30＜Super＞mRNA Purification(註冊商標)Kit(TAKARA BIO公司製造)等套組等。又，亦可使用Fast Track mRNA Isolation(註冊商標)Kit(Invitrogen公司製造)、或QuickPrep mRNA Purification(註冊商標)Kit(Pharmacia公司製造)等套組，由融合瘤細胞製備mRNA。 cDNA之合成及cDNA基因庫之製作係使用公知方法[Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)]、或SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Invitrogen公司製造)或ZAP-cDNA Synthesis(註冊商標)Kit(Stratagene公司製造)等套組等。 於製作cDNA基因庫時，作為組入以自融合瘤細胞萃取之mRNA作為模板所合成之cDNA的載體，只要為能夠組入該cDNA之載體則均可使用。例如使用ZAP Express[Strategies, 5, 58 (1992)]、pBluescript II SK(＋)[Nucleic Acids Research, 17, 9494 (1989)]、λZAPII(Stratagene公司製造)、λgt10、λgt11[DNA Cloning:A Practical Approach, I, 49 (1985)]、Lambda BlueMid(Clontech公司製造)、λExCell、pT7T3-18U(Pharmacia公司製造)、pCD2[Mol. Cell. Biol., 3, 280 (1983)]或pUC18[Gene, 33, 103 (1985)]等。 作為導入藉由噬菌體或質體載體所構建之cDNA基因庫之大腸桿菌，只要為能夠導入、表現及維持該cDNA基因庫者則均可使用。例如使用XL1-藍 MRF'[Strategies, 5, 81 (1992)]、C600[Genetics, 39, 440 (1954)]、Y1088、Y1090[Science, 222, 778 (1983)]、NM522[J. Mol. Biol., 166, 1 (1983)]、K802[J. Mol. Biol., 16, 118 (1966)]或JM105[Gene, 38, 275 (1985)]等。 自cDNA基因庫中選擇編碼非人類抗體之VH或VL之cDNA株時使用經同位素或螢光標記之探針之菌落・雜交法、或噬菌斑・雜交法[Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]等。 又，製備引子，以由mRNA合成之cDNA或cDNA基因庫作為模板，進行聚合酶聯鎖反應(Polymerase Chain Reaction)法[以下記為PCR法，Molecular Cloning, A Laboratory Manual, Second Edition , Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)]，藉此亦可製備編碼VH或VL之cDNA。 利用適宜之限制酶等將所選擇之cDNA切斷後，選殖至pBluescript SK(-)(Stratagene公司製造)等質體，藉由通常使用之鹼基序列解析方法等確定該cDNA之鹼基序列。鹼基序列解析方法係例如進行雙脫氧法[Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)]等反應後，使用ABI PRISM3700(PE Biosystems公司製造)或A.L.F.DNA核酸定序儀(Pharmacia公司製造)等鹼基序列自動分析裝置等。 根據確定出之鹼基序列而分別推測VH及VL之全胺基酸序列，與已知抗體之VH及VL之全胺基酸序列[Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]進行比較，藉此分別確認所取得之cDNA是否編碼包含分泌訊號序列之抗體之VH及VL之完全胺基酸序列。關於包含分泌訊號序列之抗體之VH及VL之完全胺基酸序列，藉由與已知抗體之VH及VL之全胺基酸序列[Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]進行比較，而可推測分泌訊號序列之長度及N末端胺基酸序列，進而可知曉該等所屬之亞群。又，關於VH及VL之各CDR之胺基酸序列，亦可藉由與已知抗體之VH及VL之胺基酸序列[Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]進行比較而發現。 又，使用所獲得之VH及VL之完全胺基酸序列，對例如SWISS-PROT或PIR-Protein等任意之資料庫進行BLAST法[J. Mol. Biol., 215, 403 (1990)]等同源性檢索，而可確認VH及VL之完全胺基酸序列是否為新穎序列。 (3)人類型嵌合抗體表現載體之構建 將分別編碼非人類抗體之VH或VL之cDNA分別選殖至(1)中所獲得之基因重組抗體表現用載體之編碼人類抗體之CH或CL之各基因之上游，藉此可構建人類型嵌合抗體表現載體。 為了將編碼非人類抗體之VH或VL之cDNA之3'末端側與人類抗體之CH或CL之5'末端側進行連結，而製作以連結部分之鹼基序列編碼適宜之胺基酸、且成為適宜之限制酶識別序列之方式設計的VH及VL之cDNA。將所製作之VH及VL之cDNA以該等以會適宜之形式表現之方式分別選殖至(1)中所獲得之基因重組抗體表現用載體之編碼人類抗體之CH或CL之各基因之上游，而構建人類型嵌合抗體表現載體。 又，亦可使用兩端具有適宜之限制酶之識別序列的合成DNA，藉由PCR法分別使編碼非人類抗體VH或VL之cDNA擴增後，選殖至(1)中所獲得之基因重組抗體表現用載體。 (4)編碼人類化抗體之V區域之cDNA之構建 編碼人類化抗體之VH或VL之cDNA可藉由如下方式構建。 分別選擇用以移植非人類抗體之VH或VL之CDR之胺基酸序列的人類抗體之VH或VL之FR之胺基酸序列。作為選擇之FR之胺基酸序列，只要為源自人類抗體者則均可使用。例如使用Protein Data Bank等資料庫中登記之人類抗體之FR之胺基酸序列、或人類抗體之FR之各亞群之共有胺基酸序列[Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]等。為了抑制抗體之結合活性之降低，選擇與原先之抗體之VH或VL之FR之胺基酸序列的同源性儘可能較高(至少60%以上)之FR之胺基酸序列。 其次，將原先之抗體之CDR之胺基酸序列分別移植至所選擇之人類抗體之VH或VL之FR之胺基酸序列中，而分別設計人類化抗體之VH或VL之胺基酸序列。考慮到抗體之基因之鹼基序列中所見之密碼子之使用頻度[Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]，而將所設計之胺基酸序列轉換成DNA序列，分別設計編碼人類化抗體之VH或VL之胺基酸序列的DNA序列。 基於所設計之DNA序列，合成長度約為100鹼基之數條合成DNA，使用該等進行PCR反應。於該情形時，考慮到PCR反應中之反應效率及能夠合成之DNA之長度，較佳為對VH、VL各設計6條合成DNA。進而，藉由對位於兩端之合成DNA之5'或3'末端導入適宜之限制酶之識別序列，可容易地將編碼人類化抗體之VH或VL之cDNA選殖至(1)中所獲得之基因重組抗體表現用載體。 PCR反應後，將擴增產物分別選殖至pBluescript SK(-)(Stratagene公司製造)等質體，藉由與(2)記載之方法相同之方法確定鹼基序列，而取得具有所需之編碼人類化抗體之VH或VL之胺基酸序列之DNA序列的質體。 或者，亦可使用基於所設計之DNA序列分別將全長VH及全長VL合成為1條長鏈DNA者代替上述PCR擴增產物。進而，藉由於合成長鏈DNA之兩端導入適宜之限制酶之識別序列，而可將編碼人類化抗體之VH或VL之cDNA容易地選殖至(1)中所獲得之基因重組抗體表現用載體。 (5)人類化抗體之V區域之胺基酸序列之改形 關於人類化抗體，若僅將非人類抗體之VH及VL之CDR移植至人類抗體之VH及VL之FR，則其抗原結合活性與原先之非人類抗體相比降低[BIO/TECHNOLOGY, 9, 266 (1991)]。關於人類化抗體，藉由鑑別人類抗體之VH及VL之FR之胺基酸序列中的直接關係到與抗原之結合之胺基酸殘基、與CDR之胺基酸殘基相互作用之胺基酸殘基、及維持抗體之立體結構並間接關係到與抗原之結合之胺基酸殘基，將該等胺基酸殘基置換為原先之非人類抗體之胺基酸殘基，從而可提高所降低之抗原結合活性。 為了鑑別關係到抗原結合活性之FR之胺基酸殘基，藉由使用X射線結晶解析[J. Mol. Biol., 112, 535 (1977)]或電腦建模[Protein Engineering, 7, 1501 (1994)]等，而可進行抗體之立體結構之構建及解析。又，針對各抗體而製作數種改形體，分別研究各者與抗原結合活性之相關性，反覆試誤，藉此可取得具有所需抗原結合活性之人類化抗體。 人類抗體之VH及VL之FR之胺基酸殘基可藉由使用改形用合成DNA進行(4)記載之PCR反應而實現改形。針對PCR反應後之擴增產物，藉由(2)記載之方法而確定鹼基序列，從而確認實施有目標改形。 (6)人類化抗體表現載體之構建 將所構建之編碼基因重組抗體之VH或VL之cDNA分別選殖至(1)中所獲得之基因重組抗體表現用載體之編碼人類抗體之CH或CL之各基因之上游，而可構建人類化抗體表現載體。 例如藉由對(4)及(5)中所獲得之構建人類化抗體之VH或VL時使用之合成DNA中之位於兩端之合成DNA之5'或3'末端導入適宜之限制酶之識別序列，而以該等以適宜形式表現之方式分別選殖至(1)中所獲得之基因重組抗體表現用載體之編碼人類抗體之CH或CL之各基因之上游。 (7)基因重組抗體之臨時表現 使用(3)及(6)中所獲得之基因重組抗體表現載體、或將該等改形而得之表現載體，進行基因重組抗體之臨時表現，而可有效率地評價所製作之多種之人類型嵌合抗體、人類化抗體之抗原結合活性。 作為導入表現載體之宿主細胞，只要為能夠表現基因重組抗體之宿主細胞，則可使用任意細胞，例如使用COS-7細胞[美國標準菌庫(ATCC，American Type Culture Collection)編號:CRL1651][Methods in Nucleic Acids Res., CRC press, 283 (1991)]。 表現載體向COS-7細胞之導入係採用DEAE-葡聚糖法[Methods in Nucleic Acids Res., CRC press (1991)]、或脂轉染法[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]等。 於導入表現載體後，培養上清液中之基因重組抗體之表現量及抗原結合活性係使用酵素免疫抗體法[Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)、單選殖抗體實驗指南、Kodansha Scientific(1987)]等進行測定。 (8)穩定表現基因重組抗體之轉形株之取得與基因重組抗體之製備 藉由將(3)及(6)中所獲得之基因重組抗體表現載體導入至適宜之宿主細胞中而可獲得穩定表現基因重組抗體之轉形株。 表現載體向宿主細胞之導入係採用電穿孔法[日本特開平2-257891號公報、Cytotechnology, 3, 133 (1990)]等。 作為導入基因重組抗體表現載體之宿主細胞，只要為能夠使基因重組抗體表現之宿主細胞則均可使用。例如使用CHO-K1(ATCC CCL-61)、DUKXB11(ATCC CCL-9096)、Pro-5(ATCC CCL-1781)、CHO-S(Life Technologies、Cat#11619)、大鼠骨髓瘤細胞YB2/3HL.P2.G11.16Ag.20(ATCC編號：CRL1662，或亦稱為YB2/0)、小鼠骨髓瘤細胞NS0、小鼠骨髓瘤細胞SP2/0-Ag14(ATCC編號：CRL1581)、小鼠P3X63-Ag8.653細胞(ATCC編號：CRL1580)、二氫葉酸還原酶基因(Dihydroforate Reductase，以下記為dhfr)缺損之CHO細胞(CHO/DG44細胞)[Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)]等。 又，亦可使用如下宿主細胞，例如α1,6-岩藻糖基轉移酶基因缺損之CHO細胞(國際公開第2005/035586號、國際公開第02/31140號)、獲得耐凝集素性之Lec13[Somatic Cell and Molecular genetics, 12, 55 (1986)]等，上述宿主細胞中之與細胞內糖核苷酸GDP-岩藻糖之合成有關的酶等蛋白質、與岩藻糖之1位α結合於N-糖苷結合複合型糖鏈之還原末端之N-乙醯葡糖胺之6位之糖鏈修飾有關的酶等蛋白質或與細胞內糖核苷酸GDP-岩藻糖向高爾基體之輸送有關的蛋白質等之活性降低或缺失。 導入表現載體後，將穩定表現基因重組抗體之轉形株於包含G418硫酸鹽(以下記為G418)等藥劑之動物細胞培養用培養基中加以培養，藉此進行選擇(日本特開平2-257891號公報)。 動物細胞培養用培養基係使用RPMI1640培養基(Invitrogen公司製造)、GIT培養基(日本製藥公司製造)、EX-CELL301培養基(JRH公司製造)、IMDM培養基(Invitrogen公司製造)或Hybridoma-SFM培養基(Invitrogen公司製造)、或對該等培養基添加有FBS等各種添加物之培養基等。藉由將所獲得之轉形株於培養基中培養而使基因重組抗體於培養上清液中表現蓄積。培養上清液中之基因重組抗體之表現量及抗原結合活性可藉由ELISA法等測定。又，可利用dhfr基因擴增系(日本特開平2-257891號公報)等而提高轉形株所產生之基因重組抗體之表現量。 基因重組抗體係使用蛋白質A-管柱由轉形株之培養上清液進行精製[Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]。又，亦可將凝膠過濾、離子交換層析及超濾等用於蛋白質精製之方法進行組合。 經精製之基因重組抗體之H鏈、L鏈或抗體分子整體之分子量可使用聚丙烯醯胺凝膠電泳法[Nature, 227, 680 (1970)]、或西方墨點法[Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]等進行測定。 3.精製單株抗體或該抗體片段之活性評價 經精製之本發明之單株抗體或該抗體片段之活性評價可藉由如下方式進行。 本發明之抗體或該抗體片段對人類CCR1之結合活性係使用上述1-(6)記載之流式細胞儀進行測定。又，可使用螢光抗體法[Cancer Immunol. Immunother., 36, 373 (1993)]等進行測定。 本發明之抗體或該抗體片段之抑制由人類CCL15引起之人類CCR1表現細胞之遷移的活性可利用上述趨化性分析而進行測定。 對人類CCR1表現細胞之CDC活性或ADCC活性可藉由公知之測定方法[Cancer Immunol. Immunother., 36, 373(1993); Current protocols in Immunology, Chapter7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons,Inc.,(1993)]進行測定。 4.控制抗體之效應器活性之方法 作為控制本發明之單株抗體之效應器活性之方法，已知對α1,6結合於N-乙醯葡糖胺(GlcNAc)之岩藻糖(亦稱為核心岩藻糖)之量加以控制之方法(國際公開第2005/035586號、國際公開第2002/31140號、國際公開第00/61739號)，上述N-乙醯葡糖胺(GlcNAc)存在於抗體之Fc區域之第297號天冬醯胺(Asn)上結合之N結合複合型糖鏈之還原末端；或藉由使抗體之Fc區域之胺基酸殘基改形而進行控制之方法等。採用上述任一方法均可控制本發明之單株抗體之效應器活性。 所謂效應器活性係指經由抗體之Fc區域而引起之抗體依賴性之活性，已知ADCC活性、CDC活性、或基於巨噬細胞或樹狀細胞等食細胞之抗體依賴性吞噬作用(Antibody-dependent phagocytosis，ADP活性)等。 作為效應器活性之測定法，例如可將作為標的之炎症性細胞、作為效應器之人類末梢血液單核球(PBMC)、及炎症性細胞特異性抗體加以混合，約培養4小時後，測定作為細胞毒性之指標的游離之乳酸脫氫酶(LDH)。或者可對人類全血添加例如識別如CD20之血液細胞特異性抗原之抗體，經培養後，測定標的血液細胞數之減少即效應器活性。或者例如可於人類全血中混合其他標的細胞，進而對標的細胞添加特異性抗體並經培養後，測定標的細胞數之減少即效應器活性。於任一情形時，效應器活性均可藉由游離LDH法、游離51
Cr法或流式細胞儀法等測定。 藉由控制抗體之Fc之N結合複合型糖鏈之核心岩藻糖之含量而可提高或降低抗體之效應器活性。作為使結合於抗體之Fc之N結合複合型糖鏈上結合之岩藻糖之含量減少的方法，使用α1,6-岩藻糖基轉移酶基因缺損之CHO細胞而表現抗體，藉此可取得未結合有岩藻糖之抗體。未結合有岩藻糖之抗體具有較高之ADCC活性。 另一方面，作為使結合於抗體之Fc之N結合複合型糖鏈上結合之岩藻糖之含量增加的方法，使用導入有α1,6-岩藻糖基轉移酶基因之宿主細胞而表現抗體，藉此可取得結合有岩藻糖之抗體。結合有岩藻糖之抗體具有低於未結合有岩藻糖之抗體之ADCC活性。 又，可藉由使抗體之Fc區域之胺基酸殘基改形而提高或降低ADCC活性或CDC活性。例如藉由使用美國專利申請公開第2007/0148165號說明書所記載之Fc區域之胺基酸序列而可提高抗體之CDC活性。 又，可藉由進行美國專利第6,737,056號說明書、美國專利第7,297,775號說明書或美國專利第7,317,091號說明書所記載之胺基酸改形而提高或降低ADCC活性或CDC活性。又，本發明之抗體亦包括如下血中半衰期經控制之抗體，該抗體係藉由對應於上述抗體恆定區中之胺基酸改形或糖鏈改形而進行例如日本專利特開第2013-165716號公報或日本專利特開第2012-021004號公報等所記載之胺基酸改形，從而控制對Fc受體之反應性，藉此控制血中半衰期。 進而，藉由對一種抗體組合使用上述方法，而可取得抗體之效應器活性或血中半衰期得到控制之抗體。 5.使用本發明之抗人類CCR1單株抗體或該抗體片段之疾病之治療方法 本發明之單株抗體或該抗體片段可用於治療只要為人類CCR1依賴性細胞遷移、病變等與CCR1有關之疾病的任意之人類CCR1相關疾病。 含有本發明之單株抗體或該抗體片段之治療劑可為僅包含作為有效成分之該抗體或該抗體片段者，但通常係以與藥理學上容許之1種以上之載體一併混合後藉由製劑學技術領域中之公知方法所製造之醫藥製劑的形式提供。 作為投予路徑，例如可列舉：經口投予、或口腔內、氣管內、直腸內、皮下、肌內或靜脈內等非經口投予。作為投予形態，例如可列舉：噴霧劑、膠囊劑、錠劑、散劑、顆粒劑、糖漿劑、乳劑、栓劑、注射劑、軟膏或貼劑等。 作為適於經口投予之製劑，可列舉：乳劑、糖漿劑、膠囊劑、錠劑、散劑或顆粒劑等。 如乳劑或糖漿劑之液體製備物係使用水、蔗糖、山梨糖醇或果糖等糖類、聚乙二醇或丙二醇等二醇類、芝麻油、橄欖油或大豆油等油類、對羥基苯甲酸酯類等防腐劑、或者草莓香料或胡椒薄荷等香料類等作為添加劑而製造。 膠囊劑、錠劑、散劑或顆粒劑等係使用乳糖、葡萄糖、蔗糖或甘露醇等賦形劑、澱粉或海藻酸鈉等崩解劑、硬脂酸鎂或滑石等潤滑劑、聚乙烯醇、羥丙基纖維素或明膠等結合劑、脂肪酸酯等界面活性劑或甘油等塑化劑等作為添加劑而製造。 作為適於非經口投予之製劑，存在注射劑、栓劑或噴霧劑等。注射劑係使用包含鹽溶液、葡萄糖溶液或該兩者之混合物之載體等而製造。栓劑係使用可可脂、氫化脂肪或羧酸等載體而製造。 噴霧劑係使用不會對受者之口腔及氣管黏膜產生刺激、且使本發明之單株抗體或該抗體片段以微細粒子之形式分散而使其易被吸收的載體等而製造。作為載體，例如使用乳糖或甘油等。又，亦可以氣溶膠或乾粉之形式製造。進而，亦可於上述非經口劑中添加針對適於經口投予之製劑所例示之作為添加劑之成分。 6.使用本發明之抗人類CCR1單株抗體或該抗體片段之疾病之診斷方法 使用本發明之單株抗體或該抗體片段，檢測或測定人類CCR1或人類CCR1表現細胞，藉此可診斷人類CCR1相關疾病。 作為人類CCR1相關疾病之癌症、自體免疫性疾病及炎症性疾病之診斷例如可藉由利用免疫學方法檢測或測定患者體內所存在之人類CCR1而進行。又，可藉由使用流式細胞儀等免疫學方法檢測患者體內之細胞中所表現之人類CCR1而進行診斷。 所謂免疫學方法係使用經標記之抗原或抗體而檢測或測定抗體量或抗原量之方法。例如使用放射性物質標記免疫抗體法、酶免疫測定法、螢光免疫測定法、發光免疫測定法、西方墨點法或物理化學方法等。 放射性物質標記免疫抗體法係例如使抗原或抗原表現細胞等與本發明之抗體或該抗體片段進行反應，進而與經放射線標記之抗免疫球蛋白抗體或該抗體片段進行反應後，利用閃爍計數器等進行測定。 酶免疫測定法係例如使抗原或抗原表現細胞等與本發明之抗體或該抗體片段進行反應，進而與經酶等標記之抗免疫球蛋白抗體或結合片段進行反應後，添加受質，利用吸光光度計而測定反應液之吸光度。例如使用三明治ELISA法等。作為酶免疫測定法中所使用之標記物，可使用公知[酶免疫測定法，醫學書院(1987)]之酵素標記。 例如使用鹼性磷酸酶標記、過氧化酶標記、螢光素酶標記或生物素標記等。三明治ELISA法係如下方法：使抗體結合於固相後，捕捉作為檢測或測定対象之抗原，使所捕獲之抗原與第2抗體反應。該ELISA法中，準備2種識別所欲檢測或測定之抗原之抗體或抗體片段，該等之抗原識別部位不同，其中，預先使第1抗體或抗體片段吸附於孔板(例如96孔板)，繼而利用FITC等螢光物質、過氧化酶等酶或生物素等標記第2抗體或抗體片段。使吸附有上述抗體之孔板與自生物體內分離出之細胞或其破碎液、組織或其破碎液、細胞培養上清液、血清、胸水、腹水或眼液等進行反應後，與經標記之單株抗體或抗體片段進行反應，進行對應於標記物質之檢測反應。根據分階段稀釋濃度已知之抗原所製作之校準曲線而算出被試驗樣品中之抗原濃度。作為三明治ELISA法中所使用之抗體，可使用多株抗體或單株抗體之任意者，亦可使用Fab、Fab'或F(ab)2
等抗體片段。作為三明治ELISA法中所使用之2種抗體之組合，可為識別不同之表位之單株抗體或抗體片段之組合，亦可為多株抗體與單株抗體或抗體片段之組合。 螢光免疫測定法係藉由文獻[Monoclonal Antibodies-Principles and practice, Third edition,Academic Press (1996)、單株抗體實驗指南、Kodansha Scientific (1987)]等中記載之方法進行測定。作為螢光免疫測定法中使用之標記物，可使用公知[螢光抗體法，soft science公司(1983)]之螢光標記。例如使用FITC或RITC等。 發光免疫測定法係藉由文獻[生物發光與化學發光 臨床檢查42，廣川書店(1998)]等中記載之方法進行測定。作為發光免疫測定法中使用之標記物，可列舉公知之發光體標記，使用吖啶鎓酯或咯吩等。 西方墨點法係利用SDS(十二烷基硫酸鈉)-PAGE(聚丙烯醯胺凝膠)[Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)]截留抗原或抗原表現細胞等後，將該凝膠轉漬(blotting)至聚偏二氟乙烯(PVDF)膜或硝化纖維素膜，使該膜與識別抗原之抗體或抗體片段進行反應，進而與經FITC等螢光物質標記、經過氧化酶等酶標記或經生物素標記等之抗小鼠IgG抗體或結合片段進行反應後，使該標記可視化，藉此進行測定。 以下，揭示一例。將表現有具有序列編號2之胺基酸序列之多肽的細胞或組織溶解，於還原條件下每條泳道以0.1～30 μg之蛋白質量藉由SDS-PAGE法進行電泳。將經電泳之蛋白質轉移至PVDF膜，與包含1～10%BSA之PBS(以下記為BSA-PBS)於室溫下反應30分鐘而進行封閉(blocking)操作。此處，使之與本發明之單株抗體反應，利用包含0.05～0.1%Tween-20之PBS(以下記為Tween-PBS)洗淨後，與經過氧化酶標記之山羊抗小鼠IgG於室溫下反應2小時。利用Tween-PBS洗淨，使用ECL Western Blotting Detection Reagents(Amersham公司製造)等檢測結合有單株抗體之區帶，藉此檢測具有序列編號2之胺基酸序列之多肽。作為利用西方墨點法進行檢測時所用之抗體，使用能夠結合於未保持天然型立體結構之多肽上之抗體。 物理化學方法例如以如下方式進行：藉由使作為抗原之人類CCR1與本發明之單株抗體或該抗體片段結合而形成凝集體，檢測該凝集體。除此以外，作為物理化學方法，亦可使用毛細管法、一維免疫擴散法、免疫比濁法或乳膠免疫比濁法[臨床檢查法提要，金原出版(1998)]等。關於乳膠免疫比濁法，若使用利用抗體或抗原致敏之粒徑0.1～1 μm左右之聚苯乙烯乳膠等載體，藉由對應之抗原或抗體而引起抗原抗體反應，則反應液中之散射光增加，透過光減少。檢測作為吸光度或積分球濁度之該變化，藉此測定被試驗樣品中之抗原濃度等。 人類CCR1表現細胞之檢測或測定可使用公知之免疫學檢測法，其中，較佳為使用免疫沈澱法、免疫細胞染色法、免疫組織染色法或螢光抗體染色法等。 免疫沈澱法係使人類CCR1表現細胞等與本發明之單株抗體或該抗體片段反應後，添加對蛋白質G-瓊脂糖凝膠等免疫球蛋白具有特異性結合能之載體而使抗原抗體複合體沈澱。或者亦可藉由如下方法進行。將上述本發明之單株抗體或該抗體片段固相化至ELISA用96孔板後，利用BSA-PBS進行封閉。於抗體為例如融合瘤培養上清液等未經精製之狀態之情形時，預先將抗小鼠免疫球蛋白、抗大鼠免疫球蛋白、蛋白質-A或蛋白質-G等固相化至ELISA用96孔板，經BSA-PBS封閉後，分注融合瘤培養上清液而使之結合。繼而，去除BSA-PBS並經PBS充分洗淨後，與人類CCR1表現細胞或組織之溶解液進行反應。利用SDS-PAGE用樣品緩衝劑自充分洗淨後之孔板萃取免疫沈澱物，藉由上述西方墨點法進行檢測。 免疫細胞染色法或免疫組織染色法係如下方法，即，根據情況為了優化抗體之通過性而利用界面活性劑或甲醇等對抗原表現細胞或組織等進行處理後，使之與本發明之單株抗體反應，進而與經FITC等螢光標記、經過氧化酶等酶標記或經生物素標記等之抗免疫球蛋白抗體或該結合片段進行反應後，使該標記可視化，於顯微鏡下進行觀察。又，可藉由螢光抗體染色法[Monoclonal Antibodies-Principles and practice, Third edition,Academic Press (1996)、單株抗體實驗指南、Kodansha Scientific (1987)]進行檢測，即，使螢光標記之抗體與細胞反應後利用流式細胞儀進行解析。尤其與人類CCR1結合之本發明之單株抗體或該抗體片段能夠藉由螢光抗體染色法而檢測出保持天然型立體結構而表現之細胞。 又，螢光抗體染色法中，於使用FMAT8100HTS系統(Applied Biosystems公司製造)等之情形時，無需將所形成之抗體-抗原複合體、與未參與形成抗體-抗原複合體之游離之抗體或抗原進行分離而即可測定抗原量或抗體量。 以下，藉由實施例而具體說明本發明，但本發明並不限定於下述實施例。 [實施例] [實施例1]人類、小鼠CCR1之表現載體之製作 (1)各CCR1基因之製作 合成下述編碼1～7之人類或小鼠之CCR1或CCR1-CCR3嵌合受體之DNA(GenScript Japan公司)。合成時附加用以組入至各載體之限制酶位點(BamHI及NotI)、與Kozak序列。 1.編碼人類CCR1(以下記為hCCR1)之cDNA序列(序列編號1) 2.編碼小鼠CCR1(以下記為mCCR1)之cDNA序列(序列編號3) 3.編碼人類CCR3(以下記為hCCR3)之cDNA序列(序列編號5) 4.編碼將人類CCR1之第1號至第31號胺基酸序列置換為人類CCR3之對應之N末端之胺基酸序列而得之嵌合受體(以下記為NC3-hCCR1)的cDNA序列(序列編號6) 5.編碼將小鼠CCR1之第1號至第31號胺基酸序列置換為人類CCR3之對應之N末端之胺基酸序列而得之嵌合受體(以下記為NC3-mCCR1)的cDNA序列(序列編號7) 6.編碼將人類CCR3之第171號至第194號胺基酸序列置換為人類CCR1之第171號至第194號胺基酸序列而得之嵌合受體(以下記為hCCR3_EL2hCCR1)的cDNA序列(序列編號8) 7.編碼將人類CCR3之第171號至第194號胺基酸序列置換為小鼠CCR1之第171號至第194號胺基酸序列所得之嵌合受體(以下記為hCCR3_EL2mCCR1)的cDNA序列(序列編號9) (2)人類CCR1表現載體之製作 利用限制酶BamHI及NotI(New England Biolab公司)處理上述(1)-1中所合成之編碼hCCR1之DNA，精製DNA片段。利用相同之限制酶處理Tol2轉位子載體(國際公開第2010/143698號)(以下記載為Tn-pMug-Hygro)，與編碼CCR1之DNA片段混合後，藉由DNA ligase(TAKARA BIO公司)處理而使之連結。將連結後之DNA導入至大腸桿菌勝任細胞(TAKARA BIO公司)，自已獲得藥劑耐性之菌落中選擇具有目標質體DNA之大腸桿菌株。再次培養該大腸桿菌株，自培養液精製轉染用DNA(以下將如此製作之質體稱為hCCR1/Tn-pMug-Hygro)。 (3)各種CCR表現載體之製作 藉由與上述(2)相同之方法使上述(1)中所合成之mCCR1、hCCR3、NC3-hCCR1、NC3-mCCR1、hCCR3_EL2hCCR1、hCCR3_EL2mCCR1與Tn-pMug-Hygro連結，構建表現載體(以下分別稱為mCCR1/Tn-pMug-Hygro、hCCR3/Tn-pMug-Hygro、NC3-hCCR1/Tn-pMug-Hygro、NC3-mCCR1/Tn-pMug-Hygro、hCCR3_EL2hCCR1/Tn-pMug-Hygro、hCCR3_EL2mCCR1/Tn-pMug-Hygro)。 (4) mCCR1表現載體之製作 藉由與上述(2)相同之方法，使上述(1)中所合成之編碼mCCR1之DNA、與對pCAGGS[Gene. 1991 Dec 15;108(2):193-9.]附加內部核糖體進入位點(IRES，internal ribosomal entry site)及新黴素耐性基因所得之載體即pCAG-IRES-neo連結，構建表現載體(以下稱為mCCR1/pCAG-IRES-neo)。 [實施例2]CCR1表現細胞株之製作 (1) hCCR1表現細胞之製作 將實施例1中所製作之作為質體DNA之hCCR1/Tn-pMug-Hygro與Tol2 transposase表現載體TPEX_pMug(國際公開第2013/005649號)共導入至CHO-S(Thermo Fisher Scientific公司)，製作表現細胞株。基因導入係使用Fugene HD(Promega公司)以如下方式進行。將製備成1×105
cell/mL之細胞以每孔2.5 mL播種至6孔板，24小時後，於培養液中添加hCCR1/Tn-pMug-Hygro、TPEX_pMug、Fugene HD之混合物。添加後72小時後添加1 mg/mL之潮黴素(Invitrogen公司)，進行約2週之藥劑選擇。回收獲得藥劑耐性之細胞，利用流式細胞儀(FACS Calibur，BD Biosciences公司)進行表現解析，結果確認到所導入之hCCR1之表現。將該細胞株稱為CHO-S-hCCR1。 (2)各種CCR表現細胞之製作 對實施例1中所製作之mCCR1/Tn-pMug-Hygro、hCCR3/Tn-pMug-Hygro、NC3-hCCR1/Tn-pMug-Hygro、NC3-mCCR1/Tn-pMug-Hygro、hCCR3_EL2hCCR1/Tn-pMug-Hygro及hCCR3_EL2mCCR1/Tn-pMug-Hygro，藉由與上述(1)相同之方法導入至CHO-S細胞，製作表現細胞株。以下將該等細胞株分別稱為CHO-S-mCCR1、CHO-S-hCCR3、CHO-S-NC3-hCCR1、CHO-S-NC3-mCCR1、CHO-S-hCCR3_EL2hCCR1及CHO-S-hCCR3_EL2mCCR1。 (3) RL33-hCCR1細胞之製作 將實施例1中所製作之hCCR1/Tn-pMug-Hygro與Tol2 transposase表現載體TPEX_pMug(國際公開第2013/005649號)共導入至兔細胞株RL-33[Yoshii et al., Jpn J Med Sci Biol. 1977 Jun;30(3):149-57]，製作hCCR1表現細胞株。基因導入係使用Lipofectamine LTX(Thermo Fisher Scientific公司)以如下方式進行。將製備成1×105
cell/mL之細胞以每孔2 mL播種至6孔板，於培養基中添加2.5 μg之質體DNA、5 μL之Lipofectamine LTX之混合物。添加後72小時後添加1 mg/mL之潮黴素，進行約2週之藥劑選擇。回收獲得藥劑耐性之細胞，利用流式細胞儀進行表現解析，結果確認到所導入之hCCR1之表現。以下將該細胞株稱為RL33-hCCR1。 (4) RL33-mCCR1細胞之製作 藉由與上述(3)相同之方法將實施例1(4)中所製作之mCCR1/pCAG-IRES-neo導入至RL-33，製作表現細胞株。藥劑選擇係利用0.5 mg/mL之G418而進行。以下將該細胞株稱為RL33-mCCR1。 [實施例3]抗CCR1兔多株抗體之製作 藉由以下之方法製作抗CCR1兔多株抗體。合成人類CCR1之N末端肽(序列編號10)，每隔2週對2隻兔(New Zealand White)各進行一次免疫，共計5次。僅於第一次免疫時使用Complete Freund's Adjuvant(CFA)，第二次以後使用Incomplete Freund's Adjuvant(IFA)，於背部之數個部位以皮下注入之方式進行。免疫後自抗體效價提高之個體採集血清，利用Protein A管柱(GE Healthcare公司)進行親和精製而精製IgG。將如此製作之抗CCR1兔多株抗體稱為E5971。 [實施例4]利用流式細胞儀之表現解析 (1) CCR1表現之確認 使用實施例3中所製作之抗CCR1兔多株抗體E5971將實施例2中所製作之CCR1表現細胞株染色，利用流式細胞儀(FCM)確認CCR1之表現。FCM解析係以如下方式進行。將細胞以2×105
cell/well播種至96孔板，利用染色緩衝液[3%FBS(Thermo Fisher Scientific公司)/DPBS(Nacalai Tesque公司)/0.1%迭氮化鈉(Nacalai Tesque公司)]洗淨。利用10 μg/mL之E5971於冰上對該細胞進行1小時之處理，經染色緩衝液洗淨後，以最終濃度1 μg/mL添加二次抗體Alexa Fluor 647 goat Anti-Rabbit IgG(Thermo Fisher Scientific公司製造)，於室溫下處理30分鐘。再次利用染色緩衝液將該細胞洗淨後，使之懸浮於染色緩衝液，使用BD FACSCalibur(BD Biosciences公司)進行解析。籍此可確認於所製作之CCR1表現細胞株中表現出所導入之CCR1。 (2) CCR3表現之確認 對實施例2中所製作之CHO-S-hCCR3亦藉由與上述(1)相同之方法確認CCR3之表現。一次抗體係使用市售之作為抗CCR3抗體之444-11抗體(MBL公司)，二次抗體係使用Alexa Fluor 647 goat Anti-mouseIgG(H＋L)(Thermo Fisher Scientific公司)。藉此可確認於CHO-S-hCCR3中表現出所導入之CCR3。 [實施例5]使用CCR1剔除小鼠之單株抗體之製作 為了獲得小鼠交叉性抗體，使用市售之CCR1剔除(KO)小鼠(B6.129S4-Ccr1tm1Gao
N10＋N5)(非專利文獻17)(Taconic公司)而製作單株抗體。抗體製作係按照如下步驟進行。 (1)免疫致敏 使用實施例2中所製作之CHO-S-hCCR1、CHO-S-mCCR1、RL33-hCCR1及RL33-mCCR1作為免疫原。每次免疫時使用1×107
細胞/隻。對5～9週齡之CCR1 KO 小鼠，僅於初次免疫時添加Alum凝膠(LSL公司)(80 μL/隻)及百日咳疫苗(Nacalai Tesque公司)(1×107
細胞/隻)作為佐劑，藉由腹腔內投予而進行免疫致敏。每次免疫時均以投予量成為500 μL/隻之方式利用PBS製備。距初次免疫經過2週後進行第2次免疫，進而於1週後進行第3次免疫，3天後進行部分採血。 (2)抗血清評價(FCM) 使用實施例2中所製作之各種CCR1表現細胞，藉由FCM測定血清中之特異性抗體效價。測定係依照以下步驟進行。利用1%BSA(Nacalai Tesque公司)-PBS(Nacalai Tesque公司)[包含0.02%EDTA(Nacalai Tesque公司)、0.05%NaN3
(Nacalai Tesque公司)]分別將細胞製備成1×105
cell/well，以50 μL/well分注至96孔細胞培養用板U底。於其中，以50 μL/well分注將作為被試驗樣品之自被免疫動物採集之血清利用1%BSA-PBS(0.02%EDTA、0.05%NaN3
)以成為最終濃度200倍稀釋、1000倍稀釋及5000倍稀釋之方式所製備者，於4℃下放置30分鐘。進行離心分離(2000 rpm，2分鐘)後，抽吸上清液，利用板振盪器使細胞之顆粒離散。以200 μL/well分注1%BSA-PBS(0.02%EDTA、0.05%NaN3
)，再次進行離心分離(2000 rpm，2分鐘)後，抽吸上清液，利用板振盪器使細胞之顆粒離散。向其中以50 μL/well分注經1%BSA-PBS(0.02%EDTA、0.05%NaN3
)製備成最終成為300倍稀釋之Alexa Fluor647 goat anti-mouse IgG(H＋L)或Alexa Fluor488 goat anti-mouse IgG(H＋L)，於遮光下4℃下放置30分鐘。進行離心分離(2000 rpm，2分鐘)後，抽吸上清液，利用板振盪器使細胞之顆粒離散。以200 μL/well分注1%BSA-PBS(0.02%EDTA、0.05%NaN3
)，再次進行離心分離(2000 rpm，2分鐘)後，抽吸上清液，利用板振盪器使細胞之顆粒離散。於其中以50 μL/well分注1%BSA-PBS(0.02%EDTA、0.05%NaN3
)，利用流式細胞儀[FACSCanto(商標)II/BD]測定螢光強度。藉此，選擇確認到抗體化提昇之個體，摘取脾臟。 (3)藉由細胞融合之融合瘤製作 利用S-Clone cloning medium(Eidia公司)培養小鼠骨髓瘤細胞株 P3-U1(P3X63Ag8U.1、ATCC CRL-1597)，經無血清適應後，用作細胞融合之母株。於無菌條件下摘取被免疫動物之脾臟，利用紅血細胞裂解緩衝液(RED BLOOD CELL LYSING BUFFER)(Sigma-Aldrich公司)使之溶血後，使用PBS清洗2次，將脾細胞與P3-U1以細胞數成為脾臟細胞：P3-U1＝8：1之方式混合，進行離心分離(1200 rpm，5分鐘)。使所獲得之沈澱部分之細胞群充分離散後，一面攪拌一面於37℃下添加1 g之聚乙二醇-1000(PEG-1000，純正化學公司)、1 mL之MEM培養基(Nacalai Tesque公司)及0.35 mL之二甲基亞碸(Sigma-Aldrich公司)之混合液0.5 mL，於其中每隔1分鐘添加1 mL之MEM培養基，添加5次後，以總量成為50 mL之方式添加MEM培養基。對細胞懸浮液進行離心分離(900 rpm，5分鐘)，緩慢地使所獲得之沈澱部分之細胞離散後，使脾臟細胞以1.5×107
cell/9 mL之細胞濃度懸浮於添加有HAT SUPPLEMENT(Thermo Fisher Scientific公司)之S-Clone cloning medium中。預先將添加有HAT之選殖培養基(cloning medium)以100 μL/well分注至96孔培養用板，於其中以100 μL/well分注細胞懸浮液，於CO2
培養箱(5%CO2
、37℃)中培養8～10天。 (4)融合瘤之篩選 藉由FCM評價融合瘤培養上清液中包含之抗體之對CCR1之結合活性。被試驗樣品係使用融合瘤培養上清液，染色與測定係藉由與上述(2)相同之步驟實施。 (5)融合瘤之次選殖 對於篩選中呈陽性之孔內之細胞進行次選殖，於選殖培養基中培養7～10天左右。 (6)抗體亞型之確定 藉由使用亞型特異性二次抗體之FCM而確定各抗體之亞型。染色、測定係藉由與上述(2)相同之步驟進行。被試驗樣品係使用融合瘤培養上清液。檢測抗體係使用Alexa Fluor488 goat anti-mouse IgG(H＋L)(Thermo Fisher Scientific公司)、及各亞型特異性抗體(Alexa Fluor488 goat anti-mouse IgG1(Thermo Fisher Scientific公司)、Alexa Fluor488 goat anti-mouse IgG2a(Thermo Fisher Scientific公司)、Alexa Fluor488 goat anti-mouse IgG2b(Thermo Fisher Scientific公司)、Alexa Fluor488 goat anti-mouse IgG3)(Thermo Fisher Scientific公司)。 (7)自融合瘤培養上清液之抗體精製 自以如上方式選殖之融合瘤之培養上清液精製抗體。精製係使用Protein G Sepharose 4Fast Flow(GE Healthcare公司)。對培養上清液進行離心分離，去除沈澱物，利用過濾器進行過濾。對管柱填充400 μL之載體，利用DPBS置換緩衝液。添加培養上清液，使抗體吸附至單體後，利用10 mL之DPBS清洗2次。添加0.4 mL之IgG Elution Buffer(Thermo Fisher Scientific公司)而溶出後，隨即利用0.1 mL之1M Tris-HCl(NIPPON GENE公司)pH值8.6進行中和。使用NAP管柱(GE Healthcare公司)進行脫鹽並進行緩衝液與DPBS之置換，用於其後之解析。將所製作之抗體之株名、來源及亞型示於表1。 [表1]
[實施例6]THP-1遷移(趨化性)分析 作為表現CCR1之人類細胞株，已知人類單核球性白血病細胞株THP-1。已知該細胞對應於CCL3、CCL5、CCL15或CCL23等CCR1配體之濃度梯度而顯示出趨化性，使用THP-1之遷移分析為通用之CCR1抑制劑之評價系統。因此，對於實施例5中所獲得之抗人類CCR1抗體，亦使用該實驗系統而評價是否抑制由人類CCL15引起之人類CCR1之活化。 以下記載遷移分析之方法。THP-1細胞獲自ATCC。將THP-1細胞於5 μM全反式視黃酸(ATRA，All-trans-retinoic acid)(和光純藥工業公司)存在下培養3天，經分化誘導後進行回收，利用加熱至37℃之分析培養基[1%FBS(Thermo Fisher Scientific公司)/RPMI1640(Nacalai Tesque公司)]洗淨後，再使之懸浮於相同之培養基中。製備成1×106
cell/mL，將細胞以100 μL/well分注至孔徑5 μm之Transwell(Corning公司，#3421)之上層。於下層注入添加有1 ng/mL之重組人類CCL15(68aa)(R＆D technologies公司，#628-LK)之分析培養基作為化學引誘劑，於37℃下在5%CO2
培養箱內培養4-6小時後，利用Celltiter-Glo(Promega公司)定量移動至下層之細胞數。 使用本測定系統而評價精製抗體之細胞遷移時，預先於1.5 mL試管內將90 μL之細胞懸浮液與10 μL之精製抗體溶液混合，於37℃下培養1小時後，將細胞分注至Trasnwell之上層。抗體係將最終濃度調整為0.3、1、3及10 μg/mL後用於測定。 將所獲得之結果示於圖1(a)及圖1(b)。如圖1(a)及圖1(b)所示，實施例5中所獲得之小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體均濃度依賴性地抑制由活化CCL15所誘導之THP-1之遷移。 根據上述情況可知，本發明之小鼠抗人類CCR1單株抗體均為抑制由人類CCL15引起之人類CCR1之活化的抗體。 [實施例7]抗人類CCR1抗體之人類CCR1結合區域之確定 使用CCR1-CCR3嵌合受體表現細胞並藉由FCM測定實施例5中所獲得之小鼠抗人類CCR1單株抗體之人類CCR1之結合區域。測定係藉由與實施例4相同之方法進行。 作為CCR1-CCR3嵌合受體表現細胞，使用實施例2中所製作之CHO-S-hCCR3、CHO-S-NC3-hCCR1、CHO-S-NC3-mCCR1、及CHO-S-hCCR3_EL2hCCR1。又，使用CHO-S作為陰性對照。 作為被試驗抗體，使用經10倍稀釋之各融合瘤培養上清液、現有之小鼠抗人類CCR1單株抗體 53504抗體(R＆D Technologies公司)及小鼠抗人類CCR3單株抗體 444-11抗體(MBL公司)。 關於測定結果，用某細胞經某被試驗抗體(各融合瘤培養上清液、53504抗體或444-11抗體)及二次抗體染色時之螢光強度除以該細胞僅經二次抗體染色時之螢光強度。於所得之數值為10以上時，判定該被試驗抗體結合於該細胞，於未達10時，判定該被試驗抗體未結合於該細胞，於表2中分別表示為○、×。 [表2]
根據表2，小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體均未結合於CHO-S-hCCR3，而結合於CHO-S-hCCR3_EL2hCCR1。因此可知，本發明之小鼠抗人類CCR1單株抗體均結合於人類CCR1之細胞外環2。 [實施例8]現有之抗人類CCR1抗體與使用抗人類CCR1抗體之趨化性分析 (1)現有之小鼠抗人類CCR1單株抗體2D4抗體之製備 自ATCC獲得可產生現有之抗人類CCR1抗體即2D4抗體(美國專利第6,756,035號說明書)的融合瘤(LS-125-2D4-11-10-1)。使用Hybridoma-SFM(Thermo Fisher Scientific公司)培養該融合瘤，自培養上清液精製抗體。精製係使用Protein G Sepharose 4Fast Flow(GE Healthcare公司)。對培養上清液進行離心分離，利用過濾器過濾所獲得之培養上清液。對管柱填充400 μL之載體，利用DPBS置換緩衝液。對該管柱添加該培養上清液，使抗體吸附於載體後，利用10 mL之DPBS清洗2次。對該管柱添加0.4 mL之IgG Elution Buffer(Thermo Scientific公司)而使抗體溶出後，隨即利用0.1 mL之1M Tris-Cl(Nacalai Tesque公司)pH值8.6中和抗體溶液。使用NAP管柱(GE Healthcare公司)進行該抗體溶液之脫鹽並進行緩衝液與DPBS之置換，用於其後之解析。 藉由通常之方法對經精製之2D4抗體進行還原條件下之SDS-PAGE，確認抗體已獲得精製。 又，依據實施例4中記載之方法並藉由FCM確認2D4抗體對人類CCR1之結合活性。2D4抗體係以0.1、1 μg/mL反應，關於細胞，使用CHO-S-hCCR1作為人類CCR1表現細胞，使用CHO-S作為陰性對照。其結果，2D4抗體未結合於CHO-S，而濃度依賴性地結合於CHO-S-hCCR1。由此可確認經精製之2D4抗體與市售之2D4抗體同樣地具有對人類CCR1之結合性。 (2)趨化性分析 針對現有之抗人類CCR1抗體、與實施例5中所獲得之小鼠抗人類CCR1抗體單株抗體KM5908抗體及KM5916抗體，依據實施例6中記載之方法測定抑制CCR1之活化之活性，將各抗體所獲得之結果進行比較。 作為現有之抗人類CCR1抗體，使用(1)中所製作之2D4抗體、141-2抗體(MBL公司)及53504抗體(R＆D Systems公司)。 將所獲得之結果示於圖2。如圖2所示，作為現有之抗人類CCR1抗體的2D4抗體、141-2抗體及53504抗體均未抑制由活化CCL15所誘導之THP-1細胞之遷移，另一方面，小鼠抗人類CCR1單株抗體KM5908抗體及KM5916抗體濃度依賴性地抑制上述細胞之遷移。 如實施例6之記載，實施例5中取得之抗人類CCR1抗體均於與本實施例相同之實驗條件下抗體濃度依賴性地抑制由活化CCL15所誘導之THP-1細胞之遷移[圖1(a)及圖1(b)]。 因此表明，現有之抗人類CCR1抗體未抑制由人類CCL15引起之人類CCR1之活化，另一方面，實施例5中所獲得之小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體均為抑制由人類CCL15引起之人類CCR1之活化的抗體。 [實施例9]基因重組抗體之製作 (1)抗體可變區基因之選殖與序列確定 使用Trizol(Life Technologies公司)自實施例5中所選殖之融合瘤萃取總RNA，藉由5'-RACE法使抗體基因擴增。RACE用cDNA之合成係使用SMARTer RACE Kit(Clontech公司)。藉由使用有對在RACE用cDNA合成過程中欲附加之序列具有特異性之引子、與小鼠Ig γ鏈或κ鏈擴增用引子(序列編號11-14)之PCR，使抗體可變區片段擴增，進行選殖，並確認該DNA片段之鹼基序列。 針對實施例5中所獲得之各抗人類CCR1抗體，將表示編碼重鏈及輕鏈之可變區之胺基酸序列之鹼基序列、根據該鹼基序列所推測之胺基酸序列及自該胺基酸序列去除訊號序列而得之胺基酸序列的序列編號分別示於表3。進而，將表示本發明之各抗體之CDR之胺基酸序列的序列編號分別示於表4。 [表3]
[表4]
(2)嵌合抗體之表現載體之製作 藉由以下記載之方法製作將實施例5中所製作之各抗人類CCR1抗體中之恆定區置換為包含S228P、L235E及R409K之胺基酸改形之人類IgG4恆定區(人類IgG4PE_R409K)而得之嵌合抗體。以選殖有(1)中所製作之編碼各抗體之可變區之胺基酸序列之鹼基序列的質體DNA作為模板，利用使用附加有相同之重組用鹼基序列之引子的PCR使編碼各抗體之可變區之胺基酸序列之鹼基序列擴增。使用In-Fusion HD Cloning Kit(Clontech公司)，使該鹼基序列與N5KG4PE R409K載體[將N5KG1載體(美國專利第6,001,358號說明書)中之編碼人類IgG1之恆定區之鹼基序列置換為編碼包含上述胺基酸改形之變異人類IgG4之恆定區之鹼基序列而得的載體(以下記載為N5KG4PE R409K載體)]連結，製作嵌合抗體之表現載體。實驗程序依據套組所隨附之指南。 (3)嵌合抗體之產生與精製 使用(2)中所製作之表現載體及Expi293 Expression System(Life Technologies公司)而產生嵌合抗體。步驟係依據隨附指南以如下方式進行。將Expi293F細胞(Thermo Fisher Scientific公司)以2×106
cell/mL之密度於37℃下培養24小時，其後以每次反應1.25×108
cell之方式添加至42.5 mL之Expi293 Expression Medium(Thermo Fisher Scientific公司)。對Opti-MEM(Thermo Fisher Scientific公司)添加50 μg之質體DNA與ExpiFectamin 293 Reagent(Thermo Fisher Scientific公司)，靜置30分鐘後，將該質體溶液添加至上述細胞液中。進而經過一晩之培養後，於該細胞液中添加ExpiFectamin 293 Transfection Enhancer(培養量總計為50 mL)。將該細胞液培養7～10天後，回收培養上清液。 抗體之精製係使用Protein G Sepharose 4Fast Flow(GE Healthcare公司)。對所回收之培養上清液進行離心分離，利用過濾器過濾所獲得之培養上清液。對管柱填充400 μL之載體，利用DPBS置換緩衝液。對該管柱添加培養上清液，使抗體吸附至單體後，利用10 mL之DPBS清洗該管柱2次。對該管柱添加0.4 mL之IgG Elution Buffer(Thermo Scientific公司)而使抗體溶出後，隨即對該抗體溶液添加0.1 mL之1M Tris-Cl pH值8.6進行中和。使用NAP管柱(GE Healthcare公司)使該抗體溶液脫鹽，用於其後之解析。 將如此所獲得之小鼠抗人類CCR1單株抗體KM5907抗體、KM5908抗體、KM5909抗體、KM5911抗體、KM5915抗體、KM5916抗體、KM5954抗體、KM5955抗體及KM5956抗體的嵌合抗體分別記載為chKM5907抗體、chKM5908抗體、chKM5909抗體、chKM5911抗體、chKM5915抗體、chKM5916抗體、chKM5954抗體、chKM5955抗體及chKM5956抗體。 [實施例10]嵌合抗體之結合性之評價 針對實施例9中所製作之嵌合抗體chKM5907抗體、chKM5908抗體、chKM5909抗體、chKM5911抗體、chKM5915抗體、chKM5916抗體、chKM5954抗體、chKM5955抗體及chKM5956抗體，依據實施例4所記載之方法，利用FCM測定對人類及小鼠CCR1之結合性。作為人類CCR1表現細胞及小鼠CCR1表現細胞，分別使用實施例2中所製作之CHO-S-hCCR1、CHO-S-mCCR1。其結果顯示chKM5955抗體結合於人類CCR1。並顯示其他嵌合抗體結合於人類及小鼠CCR1。 [實施例11]使用嵌合抗體之趨化性分析 針對實施例9中所製作之嵌合抗體chKM5907抗體、chKM5908抗體、chKM5909抗體、chKM5911抗體、chKM5915抗體、chKM5916抗體、chKM5954抗體、chKM5955抗體及chKM5956抗體，依據實施例6所記載之方法，進行抑制人類CCR1活化之活性測定。其結果顯示各嵌合抗體均抑制由活化人類CCL15引起之THP-1之遷移。 使用特定態樣而詳細地說明了本發明，但作為業者明瞭可於不脫離本發明之主旨與範圍之情況下進行各種變更及變化。再者，本申請案係基於2016年1月20日提出申請之日本專利出願(日本專利特願2016-8983號)，並引用之形式援用其全文。The present invention relates to a monoclonal antibody or a fragment of the antibody which binds to the extracellular region of human CCR1 and inhibits the activation of CCR1 caused by human CCL15. CCR1 is also known as CD191, CKR-1, HM145, Macrophage inflammatory protein 1α receptor (MIP1αR), CMKBR1 and SCYAR1. CCR1 is a GPCR having a 7-pass membrane-through structure and is a membrane protein containing 355 amino acids in total length. It is known that in a GPCR containing CCR1, a GPCR on a cell surface is activated by binding of a ligand to transmit the receptor-dependent signal to the cell, and at the same time, the calcium ion concentration in the cell increases. As a result, the cells undergo cell migration, production of chemokines, production of matrix metalloproteinase MMP, and the like. In other words, as a function of CCR1, a CCR1-dependent signal is transmitted to the cell by binding of a ligand to CCR1 on the cell surface, and at the same time, the calcium ion concentration in the cell increases. As a result, the cells undergo cell migration, the production of chemokines or the production of MMP. Examples of the ligand of human CCR1 include human CCL3, CCL5, CCL8, CCL14, CCL15, CCL16, and CCL23. Examples of the ligand for mouse CCR1 include mouse CCL3, CCL5, CCL7, and CCL9. Human CCL15 is a ligand contained in the CC chemokine family and contains 92 amino acids in total length. It is known that human CCL15 is decomposed by the action of a proteolytic enzyme to form an activated form of about 68 amino acids [hereinafter, described in the present invention as activating human CCL15 or hCCL15 (68aa)], thereby exerting a full length. CCL15 (hereinafter, described in the present invention as full-length CCL15) is more active. If human CCL15 binds to human CCR1 on the cell surface to activate the receptor, a CCR1-dependent signal is transmitted to the cell, causing activation of phospholipase C (PLC), an increase in intracellular calcium ion concentration, or nuclear. Activation of factor-κB (NF-κB). As a result, the cells undergo cell migration and the like. Examples of the antibody of the present invention include antibodies which inhibit various reactions accompanying activation of human CCR1 by human CCL15. Specific examples of the antibody of the present invention include inhibition of CCR1-dependent signal transmission selected from human CCR1-expressing cells by human CCL15, activation of PLC, increase of intracellular calcium ion concentration, activation of NF-κB, and CCR1 exhibits antibodies and the like of at least one of the migration of cells. Among them, as the antibody of the present invention, an antibody which inhibits migration of human CCR1 expressing cells induced by human CCL15 is preferred. The antibody of the present invention preferably exemplifies the above-mentioned reaction accompanying activation of human CCR1 by human CCL15, and achieves 5% or more, 10% or more, and 20% or more as compared with a control group to which only CCL15 is added without adding an antibody. 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and 90% or more of antibodies inhibited. The concentration of human CCL15 can be appropriately adjusted according to the measurement system so that the concentration of the above-mentioned reaction when human CCL15 is added becomes the maximum concentration. For example, in the method described in the examples of the present invention, the concentration of CCL15 is preferably 1 ng/mL when measuring the migration of CCR1 expressing cells. Further, the concentration of the antibody of the present invention can be appropriately adjusted depending on the measurement system. For example, in the method described in the present embodiment, when measuring the migration of CCR1 expressing cells, the concentration of the antibody of the present invention may be 0.3 μg/mL or more, preferably 1 μg/mL or more, and more preferably 3 More than μg/mL, preferably 10 μg/mL or more. In the present invention, human CCL15 may be any one of full-length CCL15 and activated human CCL15 as long as it activates CCR1. The human CCR1 expression cell may be any cell as long as it is a cell expressing human CCR1, and examples thereof include a human cell, a human cell strain, and a human CCR1 forced expression strain. Examples of human cells expressing human CCR1 include neutrophils, eosinophils, basophils, mononuclear cells, macrophages, dendritic cells, NK cells, T cells, B cells, and immature osteophytes. Ball (iMC) and osteophyte-derived suppressor cells (MDSC). The extracellular region of human CCR1 includes an N-terminal region including amino acid sequences No. 1 to 31 from the N-terminal end of the amino acid sequence of human CCR1, and includes amino acid sequences 97 to 103. Extracellular loop 1 region, extracellular loop 2 region comprising amino acid sequences 172 to 195, and extracellular loop 3 region comprising amino acid sequence 266 to 278 [Cell 72.3 (1993): 415-425] . Specific examples of the N-terminal region, the extracellular loop 1 region, the extracellular loop 2 region, and the extracellular loop 3 region include Nos. 1 to 31 and 97 to 103 of the amino acid sequence of SEQ ID NO: 2, respectively. , amino acid sequences 172-195 and 266-278. The antibody of the present invention may be any one as long as it binds to the extracellular region of the human CCR1, and is preferably at least one amine which binds to the amino acid sequence of the extracellular loop 2 region of human CCR1. An antibody to a base acid residue. Examples of the antibody include an antibody that binds to at least one amino acid residue in the amino acid sequence of Nos. 172 to 195 of the amino acid sequence of SEQ ID NO: 2. Further, the antibody of the present invention is more specifically selected from any one of the following (a) to (l). (a) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence described in SEQ ID NOs: 69, 70 and 71, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence described in SEQ ID NOs: 72, 73 and 74. (b) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 75, 76 and 77, and the amino acid sequences of CDRs 1-3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence of SEQ ID NOs: 78, 79 and 80. (c) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 81, 82 and 83, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody of the amino acid sequence of the amino acid sequence described in SEQ ID NOs: 84, 85 and 86. (d) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 87, 88 and 89, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody of the amino acid sequence of the amino acid sequence described in SEQ ID NOs: 90, 91 and 92. (e) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 93, 94 and 95, and the amino acid sequences of CDRs 1-3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence of SEQ ID NOs: 96, 97 and 98. (f) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 99, 100 and 101, and the amino acid sequences of CDRs 1-3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence of SEQ ID NO: 102, 103 and 104. (g) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 105, 106 and 107, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence of SEQ ID NOs: 108, 109 and 110. (h) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence described in SEQ ID NO: 111, 112 and 113, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence described in SEQ ID NOs: 114, 115 and 116. (i) The amino acid sequences of CDRs 1 to 3 of VH are each an amino acid sequence comprising the amino acid sequence described in SEQ ID NOs: 117, 118 and 119, and the amino acid sequences of CDRs 1 to 3 of VL are respectively included. An antibody to the amino acid sequence of the amino acid sequence described in SEQ ID NO: 120, 121 and 122. (j) an antibody which competes with at least one of the antibodies described in (a) to (i) above for binding to human CCR1. (k) an antibody that binds to an epitope comprising an epitope to which the antibody of any one of the above (a) to (i) binds. (1) An antibody that binds to the same epitope as the epitope to which the antibody of any of the above (a) to (i) is bound. The antibody of the present invention comprises an antibody having the amino acid sequences of CDRs 1-3 of VH and CDRs 1-3 of VL of the antibody, wherein the CDRs 1-3 of VH of the antibody and the amino acid sequence of CDRs 1-3 of VL are respectively The amino acid sequences of CDRs 1 to 3 of VH of any one of the antibodies (a) to (i) and CDRs 1 to 3 of VL exhibit 90% or more homology. The homology of 90% or more, specifically, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology. In the present invention, examples of the antibodies described in the above (a) to (i) include mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916. Antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody. Further, examples of the antibodies described in the above (a) to (i) include anti-human CCR1 chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, and chKM5954 antibody. , chKM5955 antibody and chKM5956 antibody. Examples of the antibody according to the above (a) to (i) include CDRs 1 to 3 of VH having the antibody of any one of the above (a) to (i), and CDRs 1 to 3 of VL. Humanized antibodies and human antibodies of amino acid sequences. The anti-system of the above (j) of the present invention is a second antibody which inhibits binding of the first antibody to human CCR1 when the antibody described in the above (a) to (i) is the first antibody. The anti-system of the above (k) of the present invention is the case where the antibody described in the above (a) to (i) is the first antibody, and the epitope to which the first antibody binds is the first epitope. Binding to a second antibody comprising a second epitope of the first epitope. Further, the anti-system of the above (1) of the present invention refers to a case where the antibody described in the above (a) to (i) is the first antibody, and the epitope to which the first antibody binds is the first epitope. At the time, the second antibody binds to the first epitope. In addition, as the antibody of the present invention, specifically, an antibody selected from any one of the following (a) to (i) may be mentioned. (a) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 51, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 52. Sequence of antibodies. (b) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 53, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 54 Sequence of antibodies. (c) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 56. Sequence of antibodies. (d) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 57, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 58 Sequence of antibodies. (e) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 59, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 60. Sequence of antibodies. (f) the amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 62 Sequence of antibodies. (g) the amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 63, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 64 Sequence of antibodies. (h) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 65, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 66. Sequence of antibodies. (i) The amino acid sequence of VH is an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 67, and the amino acid sequence of VL is an amino acid comprising the amino acid sequence of SEQ ID NO: 68. Sequence of antibodies. The antibody of the present invention includes an antibody having an amino acid sequence of VH and VL of the antibody, and the amino acid sequence of VH and VL of the antibody is respectively the same as any of the antibodies described in (a) to (i) above. The amino acid sequences of VH and VL exhibit more than 90% homology. The homology of 90% or more, specifically, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology. In the present invention, examples of the antibodies described in the above (a) to (i) include mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916. Antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody. Further, examples of the antibodies described in the above (a) to (i) include anti-human CCR1 chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, and chKM5954 antibody. , chKM5955 antibody and chKM5956 antibody. In the present invention, human CCR1 includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence of NCBI Accession No. NP_001286, or a polypeptide comprising the amine of SEQ ID NO: 2 a nucleic acid sequence or an amino acid sequence of the amino acid sequence of NCBI Accession No. NP_001286, having one or more amino acid-deleted, substituted or additional amino acid sequences, and having the function of human CCR1; or the following polypeptides, etc., comprising the sequence number The amino acid sequence described in 2 or the amino acid sequence of NCBI Accession No. NP_001286 has an amino group having a homology of 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. Acid sequence and has the function of human CCR1. A polypeptide having an amino acid sequence of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence represented by NCBI Accession No. NP_001286 can be used by using a fixed or substituted amino acid sequence. Mutation introduction method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acid Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)], for example, obtained by introducing a site-directed mutation into a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. The amount of the amino acid which is deleted, substituted or added is not particularly limited, and is preferably from 1 to several tens, for example, from 1 to 20, more preferably from 1 to several, for example, from 1 to 5 amino acids. . Examples of the gene encoding human CCR1 include the nucleotide sequence described in SEQ ID NO: 1 and the nucleotide sequence of NCBI Accession No. NM_001295. The gene encoding human CCR1 of the present invention also includes a gene comprising a DNA encoding a polypeptide comprising one or more bases deleted or substituted in the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_001295. Or an additional base sequence and having the function of human CCR1; or a gene comprising DNA encoding a polypeptide comprising at least 60% or more of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_001295 a base sequence, preferably a base sequence having 80% or more homology, more preferably a base sequence having 95% or more homology, and having the function of human CCR1; or encoding the following polypeptide The gene or the like contains the DNA which hybridizes under stringent conditions to the DNA containing the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_001295, and has the function of human CCR1. DNA which hybridizes under stringent conditions means that DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_001295 is used as a probe by colony hybridization, plaque hybridization, Southern blot hybridization or DNA that can be hybridized by DNA microarray method or the like. Specifically, a DNA which can be identified by using a DNA or a plaque derived from a hybridized colony or plaque, or a PCR product or oligo DNA having the sequence, or a slide can be cited. Hybridization at 65 ° C in the presence of 0.7 to 1.0 mol/L of sodium chloride [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University, (1995)], using 0.1 to 2 times the concentration of SSC solution (the composition of the 1X concentration of SSC solution includes 150 mmol/L sodium chloride, 15 mmol/L sodium citrate), and the filter or slide was washed at 65 ° C for identification. Examples of the DNA which can be hybridized include DNA having at least 60% or more homology with the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of NM_001295, and preferably having a homology of 80% or more. DNA, more preferably DNA having more than 95% homology. A polymorphic gene is often found in the base sequence of a gene encoding a protein of eukaryotes. The gene encoding human CCR1 of the present invention also includes a gene obtained by small-scale variation of a base sequence by the above polytype in the gene used in the present invention. The numerical value of the homology in the present invention can be a value calculated by a homology search program known to the manufacturer, except for the case where it is specifically indicated, and the base sequence can be exemplified by BLAST [J. Mol. Biol. , 215, 403 (1990)], the value calculated using the preset parameters, etc.; for the amino acid sequence, it can be enumerated in BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997), http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.htmL] The values calculated using the preset parameters, and the like. As a pre-determined parameter, when G (Cost to open gap) is a base sequence, it is 5, in the case of an amino acid sequence, when it is 11; when -E (Cost to extend gap) is a base sequence, it is 2 In the case of the amino acid sequence, the system is 1; -q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, and ‐W (wordsize) is alkali In the case of the base sequence, it is 11 residues, and in the case of the amino acid sequence, it is 3 residues; when -y[Dropoff(X) for blast extensions in bits] is in the case of blastn, it is 20, in the case of programs other than blastn 7:-X (X dropoff value for gapped alignment in bits) is 15 and -Z (final X dropoff value for gapped alignment in bits) is blastn, when the system is 50, the program other than blastn is 25 (http ://www.ncbi.nlm.nih.gov/blast/htmL/blastcgihelp.htmL). A polypeptide comprising a partial sequence of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of NCBI Accession No. NP_001286 can be produced by a method known to the manufacturer. Specifically, it can be produced by partially deleting one of the DNAs of the amino acid sequence encoding the SEQ ID NO: 2, and cultivating a transformant into which the expression vector containing the partially deleted DNA is introduced. Further, the polypeptide can be obtained by the same method as described above, and the polypeptide has an amino acid of 1 or more in the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of NCBI Accession No. NP_001286, and is substituted or substituted. Or an additional amino acid sequence. Further, the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of NCBI Accession No. NP_001286, or the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of NCBI Accession No. NP_001286 A polypeptide having 1 or more amino acid-deficient, substituted or added amino acid sequence can also be produced by a chemical synthesis method such as a mercaptomethoxycarbonyl (Fmoc) method or a third butoxycarbonyl (tBoc) method. The antibody of the present invention also includes any of a plurality of antibodies, a monoclonal antibody, and an oligo antibody. The so-called multi-strain system refers to a community of antibody molecules secreted by multi-pure antibody-producing cells. The monoclonal antibody system refers to an antibody secreted by a simple antibody-producing cell, which recognizes only one epitope (also called epitope), and the amino acid sequence (primary sequence) constituting the monoclonal antibody is uniform. The oligo-resistant system refers to a community of antibody molecules obtained by mixing a plurality of different individual antibodies. Examples of the monoclonal antibodies in the present invention include antibodies produced by fusion of tumors, and recombinant antibodies produced by transformants obtained by transformation using a expression vector containing an antibody gene. Examples of the epitope include a single amino acid sequence recognized and bound by a monoclonal antibody, a stereo structure including an amino acid sequence, an amino acid sequence modified by post-translational modification, and an amino acid containing the same. The three-dimensional structure of the sequence, and the like. The amino acid sequence obtained by modifying the post-translational modification includes an amino acid sequence in which an O-binding type sugar chain, an N-binding type sugar chain, a sulfate group or the like is bonded, and the O-binding type sugar chain-based sugar chain is bonded. In the case of Tyr and Ser having an OH substituent, the N-binding sugar chain sugar chain is bonded to have NH 2 In the case of Gln and Asn of the substituent, the sulfate-based sulfuric acid molecule is bonded to Tyr having an OH substituent. The binding of the antibody of the present invention to the extracellular region of human CCR1 can be confirmed by measuring the binding of the antibody of the present invention to human CCR1 expressing cells by ELISA, flow cytometry, and surface plasmon resonance. Further, a well-known immunological assay can also be combined [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Single Colony Antibody Experiment Guide, Kodansha Scientific (1987)] and so on. The amino acid residue or epitope of human CCR1 to which the antibody of the present invention binds can be obtained by using a defect obtained by deleting a part of human CCR1, a variant obtained by replacing a region derived from another protein, and human CCR1. A part of the peptide fragment or the like is determined by performing an antibody binding assay. Further, an antibody binding assay can also be carried out using the expression cells of the above-described defective body or variant. Further, the amino acid residue or epitope of human CCR1 to which the antibody of the present invention is bound may also be determined by adding the antibody of the present invention to a peptide fragment of human CCR1 digested with a proteolytic enzyme, and the use thereof has been used. Epitope mapping was performed by mass spectrometry. Regarding the inhibition of the activation of human CCR1 by human CCL15 by the antibody of the present invention, for example, human CCR1 can express intracellular CCR1-dependent signal transmission, activation of PLC, increase of intracellular calcium ion concentration, activation of NF-κB, Or the migration of human CCR1 expressing cells was confirmed as an indicator. Migration of cells can be measured using the chemotaxis assay described below. For example, human CCR1 expression cells are added to the upper part of the chemotaxis analysis chamber, and 1) a negative control such as a medium or DPBS, 2) human CCL15, and 3) human CCL15 and an antibody of the present invention are added to the lower part of the chamber, respectively. After incubation for a certain period of time, the number of human CCR1 expressing cells present in the lower portion of the chamber was determined by a suitable method. As a result of the obtained, when the number of cells when human CCL15 was added was increased as compared with the number of cells when the medium was added, the number of cells when human CCL15 and the antibody of the present invention were added was higher than when human CCL15 was added. When the number of cells is decreased, it can be judged that the antibody of the present invention inhibits activation of human CCR1 caused by human CCL15. Further, the inhibition of the activation of human CCR1 by human CCL15 by the antibody of the present invention can be confirmed by using the change in the intracellular calcium ion concentration of human CCR1 as an index. The change in the intracellular calcium concentration can be measured by a known method, and for example, an intracellular Ca assay kit (manufactured by Wako Co., Ltd.) or the like can be used, and the measurement can be carried out in accordance with the accompanying operation instructions. As a confirmation method, for example, changes in intracellular calcium concentration when 1) a medium or a negative control such as DPBS, 2) human CCL15, and 3) human CCL15 and an antibody of the present invention are added to human CCR1 expression cells can be measured according to the above method. If the intracellular calcium ion concentration when the human CCL15 is added is increased compared with the intracellular calcium ion concentration when the medium is added, the intracellular calcium ion concentration when the human CCL15 and the antibody of the present invention are added is added. When the concentration of intracellular calcium ions in human CCL15 is decreased, it can be judged that the antibody of the present invention inhibits the activation of human CCR1 caused by human CCL15. The antibody molecule is also called immunoglobulin (hereinafter referred to as Ig), and human antibodies are classified into isotypes of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM depending on the molecular structure. IgG1, IgG2, IgG3, and IgG4, which have relatively high homology to the amino acid sequence, are also collectively referred to as IgG. The antibody molecule system is composed of a polypeptide called a heavy chain (hereinafter referred to as an H chain) and a light chain (hereinafter referred to as an L chain). Further, the H chain is composed of VH and H chain constant regions (also referred to as CH) from the N-terminal side, and the L chain is composed of VL and L chain constant regions (also referred to as CL) from the N-terminal side. Each district is composed. CH corresponds to each subtype, and α, δ, ε, γ, and μ chains are known, respectively. CH further consists of the CH1 zone, the hinge zone, the CH2 zone, and the CH3 zone from the N-terminal side. The term "domain" refers to a functional structural unit constituting each polypeptide of an antibody molecule. Further, the CH2 region and the CH3 region are collectively referred to as an Fc region or simply as Fc. CL is known as C λ Chain and C κ chain. The CH1 region, the hinge region, the CH2 region, the CH3 region, and the Fc region in the present invention may be based on the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], according to The number of the amino acid residue at the end is specified. Specifically, CH1 is specified as the amino acid sequence of the EU index Nos. 118 to 215, the hinge is specifically the amino acid sequence of the EU index Nos. 216 to 230, and the CH2 is the amine group of the EU index Nos. 231 to 340. The acid sequence and CH3 are specifically amino acid sequences of EU index Nos. 341 to 447. The antibody of the present invention particularly includes a recombinant mouse antibody, a recombinant rat antibody, a recombinant rabbit antibody, a human type chimeric antibody (hereinafter also referred to as a chimeric antibody), and a humanized antibody produced by genetic engineering (also A gene-recombinant antibody such as a human type complementarity determining region CDR-grafted antibody) and a human antibody. The chimeric antibody means an antibody comprising VH and VL of an antibody other than a human (non-human animal), and CH and CL of a human antibody. Regarding non-human animals, any animal such as mouse, rat, hamster or rabbit can be used as long as it can produce a fusion tumor. The fusion tumor refers to a cell obtained by cell fusion of a B cell obtained from a non-human animal immune antigen and a myeloma cell derived from a mouse or the like, which produces a monoclonal antibody having a desired antigen specificity. Thus, the variable region constituting the antibody produced by the fusion tumor comprises an amino acid sequence of a non-human animal antibody. A human type chimeric antibody can be produced by, for example, obtaining a cDNA encoding VH and VL of the monoclonal antibody from a fusion tumor derived from a non-human animal cell from a monoclonal antibody, and inserting it into a human antibody encoding the same. The animal cells of the DNA of CH and CL are constructed by using a expression vector to construct a human type chimeric antibody expression vector, which is introduced into animal cells and thereby produced and expressed. The humanized anti-system refers to an antibody obtained by grafting an amino acid sequence of a CDR of a VH and VL of a non-human animal antibody into a corresponding CDR of a VH and a VL of a human antibody. The region other than the CDRs of VH and VL is referred to as a framework region (hereinafter referred to as FR). The humanized antibody can be produced by constructing a cDNA encoding an amino acid sequence of the following VH and a cDNA encoding an amino acid sequence of VL, respectively, and inserting the same into an animal having DNA encoding CH and CL of a human antibody. The cell is constructed by using a expression vector to construct a humanized antibody expression vector, which is introduced into an animal cell, and the amino acid sequence of the above VH comprises the amino acid sequence of the CDR of the VH of the non-human animal antibody and any human. The amino acid sequence of the FR of the VH of the antibody, wherein the amino acid sequence of VL comprises the amino acid sequence of the CDR of the VL of the non-human animal antibody and the amino acid sequence of the FR of the VL of any human antibody. Human antibodies originally refer to antibodies naturally present in the human body, but also include human antibody phage gene banks which have recently been produced with advances in genetic engineering, cell engineering, and engineering techniques, and gene-transgenic animals that produce human antibodies. Antibodies, etc. Human antibodies can be obtained by immunizing a mouse with a human immunoglobulin gene (Tomizuka K. et. al., Proc Natl Acad Sci US A. 97, 722-7, 2000) against the desired antigen. Further, by using a phage display gene bank obtained by amplifying an antibody gene derived from human B cells, a human antibody having a desired binding activity is selected, whereby human antibodies can be obtained without immunization ( Winter G. et. al., Annu Rev Immunol. 12: 433-55. 1994). Further, human B cells can be immortalized by using Epstein-Barr virus, thereby producing a human antibody by producing a human antibody producing a desired binding activity (Rosen A. et. al., Nature 267, 52-54. 1977). The antibody present in the human body is subjected to colonization by infecting the lymphocytes which are isolated from the blood of the human body, for example, by infecting the lymphocytes, thereby obtaining the lymphocytes which produce the antibody. The antibody is purified from the culture in which the lymphocytes are cultured. The human antibody phage gene library is realized by inserting an antibody gene prepared from human B cells into a phage gene to obtain a gene bank of a phage displaying an antibody fragment such as Fab or scFv. From the gene bank, a phage displaying an antibody fragment having a desired antigen-binding activity can be recovered by using a binding activity to an antigen to which an antigen is immobilized as an index. The antibody fragment can then be converted by genetic engineering into a human antibody molecule comprising two intact H chains and two intact L chains. A gene-transgenic animal that produces a human antibody refers to an animal that has a human antibody genome incorporated into the chromosome of a host animal. Specifically, a human antibody gene is introduced into a mouse ES cell, and the ES cell is transplanted into an early embryo of another mouse to grow it, whereby a gene-transgenic animal producing a human antibody can be produced. A method for producing a human antibody by a gene-transgenic animal which produces a human antibody, which can be obtained by a fusion tumor production method using a mammal other than a human, and cultured, thereby culturing the culture Human antibodies are produced and accumulate. The amino acid sequence of VH and VL of the antibody of the present invention may be an amino acid sequence of VH and VL of a human antibody, an amino acid sequence of VH and VL of a non-human animal antibody, or a non-human animal antibody. Any one of the VH and VL amino acid sequences of the humanized antibody obtained by grafting the CDR into the framework region of any human antibody. The amino acid sequence of CL in the antibody of the present invention may be any of an amino acid sequence of a human antibody or an amino acid sequence of a non-human animal antibody, preferably a C of an amino acid sequence of a human antibody. κ Or C λ . The CH which is an antibody of the present invention may be any type as long as it belongs to an immunoglobulin, and is preferably a subtype of IgG type, and γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4) can be used. Any of them. As the antibody of the present invention, the present invention also includes an Fc fusion protein in which an Fc is combined with an antibody fragment, and an Fc fusion protein (also referred to as an immunoadhesin) which is a combination of an Fc and a naturally occurring ligand or receptor. An Fc fusion protein obtained by fusing a plurality of Fc regions. Further, in order to keep the antibody stable and to control the half-life in blood, an Fc region or the like in which an amino acid residue is modified can also be used for the antibody of the present invention. The antibody or antibody fragment of the invention also includes an antibody comprising an amino acid residue that has been post-translationally modified. As a post-translational modification, for example, a deletion of an amino acid residue at the C-terminus of the H chain [lysine clipping] or a branamine residue at the N-terminus of the polypeptide to the bran Conversion of indoleamine (pyroGlu), etc. [Beck et al, Analytical Chemistry, 85, 715-736 (2013)]. In the present invention, the antibody fragment binds to the extracellular region of human CCR1 and inhibits antigen-binding activity of activation of human CCR1 by human CCL15. Examples of the antibody fragment of the present invention include Fab, Fab', and F(ab'). 2 , scFv, diabody, dsFv or a peptide comprising a plurality of CDRs. The Fab is in a fragment obtained by treating the IgG antibody with papain (the amino acid residue of the H chain is cleaved), and about half of the N-terminal side of the H chain is monosulfide-bonded with the L chain as a whole ( The SS bond) is obtained by binding to an antibody fragment having an antigen-binding activity of about 50,000. F(ab') 2 It is in the fragment obtained by treating IgG by gastric proteolytic enzyme (cutting at the amino acid residue of the H chain No. 234), which is slightly larger than the molecular weight of the Fab via the SS bond of the hinge region of about 100,000. An antibody fragment having antigen-binding activity. Fab' will be the above F(ab') 2 An antibody fragment having an antigen-binding activity of a molecular weight of about 50,000 obtained by cleavage of the SS bond in the hinge region. scFv is an antibody fragment having antigen-binding activity in the form of a VH-P-VL or VL-P-VH polypeptide obtained by linking one VH to one VL using a suitable peptide linker (P), the above-mentioned peptide linker (P) is a linker peptide in which a linker (G4S) of four Gly and one Ser residue is connected by an arbitrary number. Diabody is an antibody fragment obtained by forming a dimer from an scFv having the same or different antigen binding specificity and having a bivalent antigen binding activity to the same antigen or having specific antigen binding activity to different antigens. dsFv refers to a polypeptide obtained by replacing one amino acid residue of VH and VL with a cysteine residue, respectively, via an SS bond between the cysteine residues. The peptide comprising the CDRs comprises at least one region of the CDRs of VH or VL. Peptides comprising a plurality of CDRs can bind the CDRs directly to each other or via a suitable peptide linker. The DNA encoding the CDRs of VH and VL of the modified antibody of the present invention can be constructed, and the DNA can be inserted into a expression vector for prokaryote or a expression vector for eukaryote, and the expression vector can be introduced into a prokaryote or eukaryote. In this way, it is made to be produced. Further, the peptide containing the CDR can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method. The monoclonal antibody of the present invention includes a monoclonal antibody that binds to human CCR1 of the present invention, or chemically or genetically engineers the antibody fragment with a radioisotope, a low molecular drug, a polymer drug, a protein or an antibody drug, and the like. A derivative of an antibody obtained by binding. A derivative of an antibody can be obtained by a chemical method [Introduction to Antibody Engineering, Takusho (1994)], which binds to the monoclonal antibody of human CCR1 of the present invention or the N-terminal side of the H chain or the L chain of the antibody fragment, The C-terminal side, a suitable substituent in the antibody molecule, or a side chain or a sugar chain are produced in combination with a radioisotope, a low molecular drug, a polymer drug, an immunostimulating agent, a protein, an antibody drug, or a nucleic acid drug. Furthermore, it can also be produced by a genetic engineering method in which a DNA encoding a monoclonal antibody or a human antibody which binds to human CCR1 of the present invention is linked to a DNA encoding a protein to be bound or an antibody medicine. Inserted into a performance vector, the expression vector is introduced into a suitable host cell for expression. As the radioactive isotope, for example, 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 At and so on. The radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance chelated with a radioisotope may be bound to the antibody. Examples of the chelating agent include 3-methyldiethylethaminepentaacetate 1-isothiocyanatobenzyl ester (MX-DTPA). Examples of the low molecular weight agent include an alkylating agent, a nitrosourea agent, a metabolic antagonist, an antibiotic, a plant base, a topoisomerase inhibitor, a hormone therapy agent, a hormone antagonist, and an aromatase inhibitor. Anticancer agents such as P glycoprotein inhibitors, platinum complex derivatives, M phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], Hydrocortisone or Prednisone, etc. Non-steroidal agents such as steroids, aspirin or indomethacin, immunomodulators such as sodium thiomalate or penicillamine, immunosuppressants such as cyclophosphamide or azathioprine or chlorpheniramine maleate Or anti-inflammatory agents such as antihistamines such as clemastine [inflammation and anti-inflammatory therapy, Medical Tooth Pharmaceutical Publishing Co., Ltd. (1982)], and the like. Examples of the anticancer agent include amifostine (Ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, and mechlorethamine/nitrogen mustard. Streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), rutinum (CCNU), doxorubicin (adremycin), epirubicin , gemcitabine (Gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, periwinkle Alkali, vincristine, bleomycin, daunomycin, pilomycin, estramustine, taxol (Taxo), taxol (Taxotere), aldesleukin (aldesleukin), aspartame Aminase (asparaginase), busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38) ), Fluxuridine, Fludarabine, Hydroxyurea, Edaby Star, Mesna, Irinotecan (CPT-11), Topotecan, Mitoxantrone Topology Topotecan, Leuprolide, megestrol, melphalan, hydrazine, hydroxyurea, plicamycin, mitotane, and parmozyme (pegaspargase), pentostatin, pipobroman, tamoxifen, goserelin, leuprorelin, flutamide, teniposide, testicular lactone ( Testolactone), thioguanine, thiotepa, uracil mustard, vinorelbine, Chlorambucil, hydrocortisone, prednisolone, methylprednisone Dragon, vindesine, nimustine, semustine, capecitabine, leutriprex, azacitidine, UFT, oxaloplatin, gemifloxacin (Iressa )), imatinib (STI571), erlotinib, FMS-like tyrosine kinase 3 inhibitor, vascular endothelial growth facotr receptor (VEGFR) Inhibitor, fibroblast growth factor receptor (FGFR) inhibitor, Iressa or Tarceva and other epidermal growth factor receptor (EGFR) inhibitors, Radicicol, 17-allylaminos-17-desmethoxylatrine , rapamycin, amsacrine, all-trans retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, Gold thiomalate, D-penicillamine, Bucillamine, azathioprine, imidazoribine, cyclosporine, rapamycin, hydrocortisone, beserrotene (Targretin)), tamoxifen, dexamethasone, progesterone, estrogen, anastrozole (Arimidex), lycopene (leuplin), aspirin, indomethacin, seléco Icta, penicillamine, sodium thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine, vitamin A acid, beserrotene, arsenic, bortezomib, allopurinol , calicheamicin, Ibritumomab tiuxetan, Targretin, ozogamine, Clarithromycin, formazan tetrahydrofolate (Le Ucorovin), Ketoconazole, Aminoglutethimide, Suramin or maytansinoid or derivatives thereof. Examples of the method for binding a low molecular drug to an antibody include a method of binding a drug to an amine group of an antibody via glutaraldehyde, or an amine group and an antibody of a drug via a water-soluble carbodiimide. a method of bonding a carboxyl group or the like. Examples of the polymer agent include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinylpyrrolidone, and pyran. Copolymer, or hydroxypropylmethacrylamide or the like. By combining these polymer compounds with an antibody or the antibody fragment, (1) stability of various chemical, physical or biological factors can be expected, (2) blood half-life is significantly prolonged, or (3) Effect of disappearance of immunogenicity or inhibition of antibody production [Bio-composite pharmaceutical products, Hirokawa Bookstore (1993)]. For example, a method of reacting PEG with an antibody may be exemplified by a method of reacting with a PEGylation-modifying reagent, etc. [Bio-composite pharmaceutical product, Hirokawa Shoten (1993)]. Examples of the PEGylation-modifying agent include a modifier for an epsilon-amino group of an lysine (Japanese Patent Laid-Open Publication No. SHO 61-178926), and a modifier for a carboxyl group of aspartic acid and glutamic acid ( Japanese Laid-Open Patent Publication No. SHO 56-23587, or a modifier for a thiol acid thiol group (Japanese Laid-Open Patent Publication No. Hei-2-17920). The immunostimulating agent may be a natural product known as an immunoadjuvant, and specific examples thereof include β(1→3) glucan (for example, lentinan or sesame (Sizofiran)) as an immunopotentiating agent. Α-galactosyl-based ceramide (KRN7000) or the like. Examples of the protein include a cytokine or a growth factor or a toxin protein which activates an immunologically active (suitable) cell such as NK cells, macrophages or neutrophils. Examples of the cytokine or growth factor include interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-12, and IL-15. IL-18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF), etc. . Examples of the toxin protein include ricin (Ricin), diphtheria toxin, and ONTAK, and protein toxins obtained by introducing a mutation into a protein to regulate toxicity. Examples of the antibody medicine include an antibody corresponding to an antigen, that is, an antigen that induces apoptosis by binding to an antibody, an antigen associated with the formation of a tumor, an antigen that regulates an immune function, or an antigen associated with an angiogenesis of a lesion. . Examples of the antigen that induces apoptosis by binding to an antibody include a cluster of differentiation (hereinafter referred to as CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II) or Epidermal Growth Factor Receptor (EGFR). Examples of the antigen related to the pathogenesis of the tumor or the antigen regulating the immune function include CD4, CD40, CD40 ligand, and B7 family molecules (for example, CD80, CD86, CD274, B7-DC, B7-H2, B7-). H3 or B7-H4), ligands of the B7 family of molecules (eg CD28, CTLA-4, ICOS, PD-1 or BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF, tumor necrosis) Factor) receptor family molecules (eg, DR4, DR5, TNFR1, or TNFR2), TNF-related apoptosis-inducing ligand receptor family molecules, receptor families of TRAIL family molecules (eg, TRAIL) -R1, TRAIL-R2, TRAIL-R3 or TRAIL-R4), receptor factor activator of nuclear factor kappa B ligand, RANK ligand, CD25, folate receptor, cytokine [eg IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF), or TNFα, or the cytokines a steroid, or a chemokine (eg, SLC, ELC, I-309, TARC, MDC, or CTACK, etc.) or a receptor for such chemokines. Examples of the antigen of an antibody that inhibits angiogenesis at a lesion site include vascular endothelial growth factor (VEGF), angiopoietin (antiopoietin), fibroblast growth factor (FGF), and EGF. Hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), erythropoietin (EPO), TGFβ , IL-8, ephrin or SDF-1 or such receptors. For the fusion antibody with a protein or antibody medicine, a cDNA encoding a monoclonal antibody or an antibody fragment can be ligated to a cDNA encoding an antibody or an antibody contained in an antibody medicine, and a DNA encoding the fusion antibody can be constructed, and the DNA can be inserted into a prokaryote or The expression vector for eukaryotes is introduced into a prokaryote or a eukaryote to thereby produce a fusion antibody. Examples of the nucleic acid medicine include pharmaceuticals including nucleic acids such as small interference ribonucleic acid (siRNA) or microRNAs (microRNAs) which act on a living body by controlling the function of a gene. For example, a complex with a nucleic acid drug that inhibits the main transcription factor RORγt of Th17 cells is conceivable. When the derivative of the antibody of the present invention is used for the detection and measurement of human CCR1 and the diagnosis of a human CCR1-related disease, the agent to be bound to the antibody may be used in a general immunological test or assay. Mark. Examples of the label include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or oxopent, or a fluorescent isothiocyanate (FITC) or isothiocyanate. Fluorescent substances such as tetramethylrhodamine (RITC). Further, the present invention includes a composition comprising a monoclonal antibody that binds to human CCR1 or the antibody fragment as an active ingredient. Further, the present invention relates to a therapeutic agent for a human CCR1-related disease, which comprises a monoclonal antibody or a antibody fragment which binds to human CCR1 as an active ingredient. Further, the present invention relates to a method for treating a human CCR1-related disease which comprises administering a monoclonal antibody or a fragment of the antibody which binds to human CCR1. The human CCR1-related disease may be any disease associated with a ligand of human CCR1 or human CCR1, and examples thereof include cancer, autoimmune diseases, and inflammatory diseases. Examples of the cancer include diffuse large cell type B cell lymphoma, filtering cell lymphoma, B cell lymphoma, T cell lymphoma, plasma cell myeloma, acute osteosarcoma, Hodgkin's lymphoma, Chronic lymphocytic leukemia, hairy cell leukemia, mantle cell lymphoma, medullary lymphoma, small lymphocytic lymphoma, multiple myeloma, hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, oral squamous cell carcinoma, Ovarian cancer, prostate cancer, breast cancer, glioma or osteosarcoma. Examples of autoimmune diseases or inflammatory diseases include rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, lupus nephritis, asthma, atopic dermatitis, and inflammatory large intestine. Inflammation, Crohn's disease or Beth's disease. The therapeutic agent containing the antibody of the present invention or the antibody fragment may be one containing only the antibody or the antibody fragment as an active ingredient, but it is preferably mixed with one or more kinds of carriers which are usually pharmacologically acceptable. It is provided in the form of a pharmaceutical preparation made by any of the methods known in the art of formulation. The administration route is preferably the most effective when the treatment is used, and examples thereof include oral administration, oral administration, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration, preferably intravenous. Invested internally. Examples of the administration form include a spray, a capsule, a tablet, a powder, a granule, a syrup, an emulsion, a suppository, an injection, an ointment, a patch, and the like. The amount of administration or the number of administrations varies depending on the target therapeutic effect, administration method, treatment time, age, and body weight, and is usually 10 μg/kg to 10 mg/kg per day for adults. The present invention relates to a method for detecting or measuring CCR1 comprising a monoclonal antibody or a human antibody that binds to human CCR1, or a method for detecting or measuring CCR1 using a monoclonal antibody or a monoclonal antibody that binds to human CCR1. In the present invention, as a method for detecting or measuring human CCR1, any known method can be mentioned. For example, an immunological detection or measurement method can be mentioned. The immunological detection or measurement method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or an antibody. Examples of the immunological detection or measurement method include a radioactive substance labeled immunological antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a fluorescent immunoassay (FIA), and a luminescent immunoassay. Western dot method or physicochemical method. The present invention relates to a diagnostic agent for a CCR1-related disease, which comprises a monoclonal antibody or a fragment thereof which binds to human CCR1, or a diagnostic method for a CCR1-related disease, which comprises using a monoclonal antibody that binds to human CCR1 or The antibody fragment is used to detect or measure CCR1. By using the monoclonal antibody or the antibody fragment of the present invention, human CCR1 expressing cells can be detected or measured according to the above method, and a disease associated with human CCR1 can be diagnosed. In the present invention, the biological sample to be used for detecting or measuring human CCR1 is not particularly limited as long as it may include human CCR1 or human CCR1 expressing cells, such as tissues, cells, blood, plasma, serum, Pancreatic juice, urine, feces, tissue fluid or culture fluid. The diagnostic agent containing the monoclonal antibody of the present invention or the antibody fragment may include a reagent for performing an antigen-antibody reaction and a reagent for detecting the reaction according to a target diagnostic method. Examples of the reagent for performing the antigen-antibody reaction include a buffer, a salt, and the like. Examples of the reagent for detection include a reagent for identifying a monoclonal antibody or a labeled secondary antibody of the antibody fragment, or a normal immunological detection or assay such as a substrate corresponding to the label. Further, the present invention relates to an anti-human CCR1 monoclonal antibody or the use of the antibody fragment for the manufacture of a therapeutic or diagnostic agent for a CCR1-related disease. Hereinafter, the method for producing the antibody of the present invention, the method for treating the disease, and the method for diagnosing the disease will be specifically described. 1. Method for producing antibody (1) Preparation of antigen Human CCR1 or human CCR1 expressing cells which can be antigens can be introduced into Escherichia coli, yeast, insect cells or animals by expressing a vector containing cDNA encoding the full length or partial length of human CCR1. Obtained in cells and the like. Further, human CCR1 can also be obtained by refining human CCR1 from various human cell strains, human cells, and human tissues expressing human CCR1 in large amounts. Further, these human cell strains, human cells, human tissues, and the like can be directly used as antigens. Further, a synthetic peptide having a partial sequence of human CCR1 can also be produced by an chemical synthesis method such as Fmoc method or tBoc method for use in an antigen. For human CCR1 or a synthetic peptide having a partial sequence of human CCR1, a known label such as FLAG or His may be added to the C-terminus or the N-terminus. The human CCR1 used in the present invention may be a method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), and the like. For example, the DNA encoding the human CCR1 is expressed in a host cell by the following method. First, a recombinant vector is produced by inserting a full-length cDNA comprising a portion encoding human CCR1 downstream of a promoter of a suitable expression vector. Instead of the above full-length cDNA, a DNA fragment of an appropriate length comprising a portion encoding the polypeptide prepared based on the full-length cDNA can also be used. Next, the obtained recombinant vector is introduced into a host cell suitable for the expression vector, whereby a polypeptide for producing a polypeptide can be obtained. As the expression vector, any promoter can be used as long as it can autonomously replicate in the host cell to be used or can be incorporated into a chromosome and contains a suitable promoter at the position where the DNA of the polypeptide can be transcribed. The host cell can be used as long as it is a microorganism, a yeast, an insect cell, or an animal cell, such as Escherichia, which can express a target gene. In the case of using a prokaryote such as Escherichia coli as a host cell, the recombinant vector is preferably capable of autonomous replication in a prokaryote and includes both a promoter, a ribosome binding sequence, a DNA comprising a portion encoding human CCR1, and a transcription termination sequence. a. Further, for the recombinant vector, the transcription termination sequence is not essential, but it is preferred to arrange a transcription termination sequence immediately downstream of the structural gene. Furthermore, the recombinant vector may also comprise a gene that controls the promoter. As the recombinant vector, a plastid which is adjusted to an appropriate distance (for example, 6 to 18 bases) between a Shine-Dalgarno sequence (also referred to as an SD sequence) as a ribosome binding sequence and a start codon is preferably used. . Further, the base sequence encoding the DNA of the human CCR1 can be substituted for the base so as to be the most suitable codon for expression in the host, whereby the productivity of the target human CCR1 can be improved. The performance vector can be used as long as it can function in the host cell to be used, and examples thereof include pBTrp2, pBTac1, pBTac2 (above, manufactured by Roche Diagnostics Co., Ltd.), pKK233-2 (manufactured by Pharmacia Co., Ltd.), and pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega Corporation), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Patent Laid-Open Publication No. SHO 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984) )], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (Manufactured by Stratagene) ), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), JP-A-60-221091, pGKA2 [Prepared by Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Laid-Open Publication No. SHO 60-221091], pTerm2 (U.S. Patent No. 4,686,191, U.S. Patent No. No. 4,939,094, US Patent No. 160,735, pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), or pME18SFL3. The promoter may be any one as long as it can function in the host cell to be used. For example, a promoter derived from Escherichia coli or bacteriophage, such as a trp promoter (Ptrp), a lac promoter, a PL promoter, a PR promoter, or a T7 promoter, may be mentioned. Further, for example, a promoter which is artificially designed and modified, such as a tandem promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, or a let I promoter, etc., may be mentioned. Examples of the host cell include Escherichia coli XL1-blue, Escherichia coli XL2-blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, E. coli NY49 or Escherichia coli DH5α. The method of introducing the recombinant vector into the host cell can be used as long as it is a method of introducing DNA into the host cell to be used, and for example, a method using calcium ions can be cited [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)]. In the case of using an animal cell as a host, the expression vector can be used as long as it can function in an animal cell, and examples thereof include pcDNAI, pCDM8 (manufactured by Funakoshi Co., Ltd.), and pAGE107 [Japanese Patent Laid-Open No. 3- Japanese Patent Publication No. 22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. Hei 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (Invitrogen), pcDNA3 .1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (International Publication No. 97/10354), N5KG1val (US Patent No. 6,001,358) No. specification), INPEP4 (manufactured by Biogen-IDEC Co., Ltd.), and transposon carrier (International Publication No. 2010/143698) and the like. The promoter can be used as long as it can function in animal cells, and examples thereof include a promoter of an immediate early (IE) early megalovirus (CMV) gene, an initial promoter of SV40, and Promoter of retrovirus, metallothionein promoter, heat shock promoter, SRα promoter or promoter or promoter of Moloney mouse leukemia virus. Further, the promoter of the IE gene of human CMV can also be used together with a promoter. Examples of the host cell include human leukemia cell Namalwa cell, monkey cell COS cell, and Chinese hamster ovary cell CHO cell [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883- 900)]; CHO cells (CHO/DG44 cells) deficient in the dihydrofolate reductase gene (hereinafter, referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 ( ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3, rat myeloma cells YB2/3HL.P2 .G11.16Ag.20 (or YB2/0), mouse myeloma cell NSO, mouse myeloma cell SP2/0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese Patent Laid-Open No. 63-000299) Bulletin) and so on. The method of introducing the recombinant vector into the host cell can be used as long as it is a method of introducing DNA into the animal cell, and examples thereof include an electroporation method [Cytotechnology, 3, 133 (1990)], and a calcium phosphate method (Japanese special) Kaiping No. 2-227075 or lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like. By obtaining a microorganism or an animal cell or the like which retains a recombinant vector encoding DNA of human CCR1 as described above, a transformant derived from the microorganism or animal cell or the like is cultured in a medium to generate the human CCR1 in the culture solution. It is continuously accumulated and collected from the culture solution, thereby producing human CCR1. The method of culturing the transformant in the medium can be carried out according to the usual method used for the culture of the host. In the case of performance in cells derived from eukaryotes, human CCR1 to which a sugar or a sugar chain is attached can be obtained. When a microorganism transformed with a recombinant vector using an inducible promoter is cultured, an inducer may be added to the medium as needed. For example, when culturing a microorganism transformed with a recombinant vector using a lac promoter, isopropyl-β-D-thiogalactoside may be added to the culture medium, and the use of the trp promoter may be employed. When the recombinant vector is cultured by the transformed microorganism, hydrazine acrylic acid or the like may be added to the culture medium. As a medium for cultivating a transformant obtained by using an animal cell as a host, for example, a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] or Iscove modified Dürr A medium such as IMsco (Iscove's Modified Dulbecco's Medium) or a medium such as fetal bovine serum (FBS) is added to the medium. Culture is usually at pH 6-8, 30-40 °C, 5% CO 2 It is carried out for 1 to 7 days under the conditions of the presence. Further, during the culture process, an antibiotic such as clarithromycin or penicillin may be added to the culture medium as needed. As a method for expressing a gene encoding human CCR1, for example, in addition to direct expression, methods such as secretion production or fusion protein expression may be cited [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]. As a production method of human CCR1, for example, a method of production in a host cell, a method of secretion to a host cell, or a method of production on an outer membrane of a host cell can be carried out by changing the host cell used or to produce The appropriate method is chosen for the structure of human CCR1. When human CCR1 is produced in a host cell or on the outer membrane of a host cell, it can be carried out by using the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], Laura et al. [Proc. The method described in Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), Japanese Patent Laid-Open Publication No. Hei 05-336963, or International Publication No. 94/23021, And human CCR1 is actively secreted outside the host cell. In addition, the production amount of human CCR1 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (JP-A No. 2-227075). The obtained human CCR1 can be isolated and purified, for example, by the following means. When human CCR1 is expressed in a dissolved state in a cell, after the completion of the culture, the cells are recovered by centrifugation, suspended in an aqueous buffer, and then an ultrasonic crusher, a fresh press, or the like. A cell-free extract is obtained by disrupting cells such as a Manton-Gaulin homogenizer or a bead mill. The supernatant can be obtained by centrifuging the cell-free extract, and the usual protein can be used alone or in combination, that is, a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, and a utilization. Precipitation method of organic solvent, anion exchange chromatography using resin such as diethylaminoethyl (DEAE)-Sepharose (Sepharose), DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), using S-Sepharose Cation exchange chromatography of resin such as FF (manufactured by Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose or phenyl sepharose, gel filtration using molecular sieve, affinity chromatography A method such as chromatofocusing or electrophoresis such as electrophoresis is used to obtain a purified sample from the supernatant. In the case where human CCR1 is expressed in the form of insolubles in the cells, the cells are recovered by the same manner as described above, and then disrupted, and centrifuged to recover the insoluble body of the human CCR1 as a precipitate. The recovered human CCR1 insoluble matter is solubilized by a protein modifier. After the human CCR1 is restored to a normal three-dimensional structure by diluting or dialyzing the solubilizing solution, a purified sample of the polypeptide can be obtained by the same separation purification method as described above. When a derivative such as human CCR1 or a glycoform thereof is secreted outside the cell, a derivative such as human CCR1 or a glycoform thereof can be recovered from the culture supernatant. The culture can be treated by a method such as centrifugation in the same manner as described above to obtain a soluble fraction, and a purified specimen can be obtained from the soluble fraction by using the same separation purification method as described above. Further, the human CCR1 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method. Further, chemical synthesis can be carried out by a peptide synthesizer manufactured by Advanced Chemtec, manufactured by Perkin Elmer, manufactured by Pharmacia, manufactured by Protein Technology Instrument, manufactured by Synthecell-Vega, manufactured by Per Septive, or manufactured by Shimadzu Corporation. (2) Preparation of antibody-immunized cells for immunization and fusion For animals such as mice, rats or hamsters 3 to 20 weeks old, the antigen obtained in (1) is used for immunization from the spleen and lymph nodes of the animal. Antibody-producing cells are collected from peripheral blood. Alternatively, mouse CCR1 knockout mice can also be used as the immunized animal. The immune system is carried out by administering the antigen to the subcutaneous, intravenous or intraperitoneal cavity of the animal together with a suitable adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel or pertussis vaccine. In the case where the antigen is a partial peptide, a complex with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet hemocyanin) is prepared and used as an immunogen. The administration of the antigen is carried out 5 to 10 times at intervals of 1 to 2 weeks after the first administration. Blood was collected from the fundus venous plexus on the 3rd to 7th day after each administration, and the antibody titer of the serum was measured using an enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum exhibits sufficient antibody titer for the antigen used for immunization is used as a supply source of the antibody-producing cells for fusion. On the 3rd to 7th day after the final administration of the antigen, the tissue containing the antibody-producing cells such as the spleen is extracted from the immunized animal, and the antibody-producing cells are collected. In the case of using spleen cells, the spleen is chopped and dispersed, and then centrifuged to remove red blood cells to obtain antibody-producing cells for fusion. (3) Preparation of myeloma cells As myeloma cells, strain cells obtained from mice are used, for example, 8-nitroguanine-resistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-U1 ( P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2/0- Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256 , 495 (1975)] and so on. The myeloma cells were subcultured in normal medium [RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine], 3 to 4 days before cell fusion. Subculture to normal medium, ensuring 2 × 10 cells on the day of fusion 7 More than one. (4) Preparation of cell fusion and monoclonal antibody-producing fusion tumor The antibody-producing cells obtained in (2) and the myeloma cells obtained in (3) were cultured in MEM (Minimum Essential Medium) medium. Or PBS (1.83 g of disodium hydrogen phosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of salt, 1 L of distilled water, pH 7.2), and fully wash the cells with the number of cells as fusion: myeloma cells = 5 to 10 The mixture was mixed in a manner of 1:1, and after centrifugation, the supernatant was removed. After the precipitated cell population was sufficiently dispersed, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium, and dimethyl hydrazine was added thereto while stirring at 37 °C. Further, 1 to 2 mL of MEM medium was added every 1 to 2 minutes, and after several additions, the MEM medium was added so that the total amount became 50 mL. After centrifugation, the supernatant was removed. After the precipitated cell population was slowly dispersed, the antibody-producing cells for fusion were slowly suspended in HAT medium [normal medium supplemented with hypoxanthine, thymidine, and amine guanidine]. The suspension was at 5% CO 2 Incubate at 37 ° C for 7 to 14 days in an incubator. After the culture, a part of the culture supernatant is taken, and a cell population which reacts with an antigen containing human CCR1 and does not react with an antigen containing no human CCR1 is selected by the following method of selection of fusion tumors such as binding assay. Next, the colonization was carried out by the limiting dilution method, and those who stably confirmed the strong antibody titer were selected as monoclonal antibodies to produce fusion tumors. (5) Preparation of purified monoclonal antibody 8 to 10 weeks after treatment with decane [intraperitoneal administration of 2,6,10,14-tetramethylpentadecane (Pristane 0.5 mL) for 2 weeks] In vitro mice or nude mice, the monoclonal antibodies obtained by intraperitoneal injection (4) produce fusion tumors. After 10 to 21 days, the ascites became cancerous in the fusion tumor. Ascites was collected from the mouse, centrifuged, and the solid content was removed, and then salted out with 40-50% ammonium sulfate by octanoic acid precipitation, DEAE-Sepharose column, protein A-column or coagulation. The gel filtration column was purified, and IgG or IgM fraction was collected as a purified monoclonal antibody. Further, the monoclonal antibody-producing fusion tumor obtained in (4) may be cultured in RPMI1640 medium supplemented with 10% FBS, and then the supernatant may be removed by centrifugation to suspend it in the fusion tumor SFM. The medium is cultured for 3 to 7 days. The obtained cell suspension was centrifuged, and the obtained supernatant was purified by a protein A-column or a protein G-column, and an IgG fraction was collected to obtain a purified monoclonal antibody. Furthermore, 5% Daigo's GF21 can also be added to the fusion tumor SFM medium. The subtype of antibody is determined using a subtype typing kit and by enzyme immunoassay. The quantification of protein quality was calculated according to the Lowry method or the absorbance at 280 nm. (6) The selection of the monoclonal antibody is as follows. The selection of the monoclonal antibody is carried out by measuring the binding of the antibody to human CCR1 expressing cells by using a flow cytometer. The human CCR1 expression cell may be any cell which expresses human CCR1 on the cell surface, and examples thereof include a human cell, a human cell strain, and a human CCR1 forced expression cell strain obtained in (1). After the human CCR1 expression cells are dispensed into a culture plate such as a 96-well plate, the test substance such as the serum of the first antibody, the culture supernatant of the fusion tumor, or the purified monoclonal antibody is dispensed and reacted. The cells after the reaction are sufficiently washed with PBS (hereinafter referred to as BSA-PBS) containing 1 to 10% bovine serum albumin (BSA), and then a fluorescent reagent such as a second antibody is dispensed. The anti-immunoglobulin antibody is labeled to react. After sufficiently washing with BSA-PBS or the like, the amount of fluorescence of the labeled antibody is measured by a flow cytometer, thereby selecting a monoclonal antibody that specifically reacts with human CCR1 expressing cells. Further, an antibody which competes with the antibody of the present invention for binding to human CCR1 can be obtained by adding and reacting the test antibody to the above-described measurement system using a flow cytometer. That is, an antibody which inhibits the binding of the antibody of the present invention to human CCR1 when the antibody to be tested is added is selected, whereby an amino acid sequence which competes with the antibody of the present invention for binding to the amino acid sequence of human CCR1 or a smectic structure thereof can be obtained. antibody. Further, an antibody which binds to an epitope comprising an epitope to which a monoclonal antibody to which human CCR1 binds in the present invention binds can be obtained by identifying a method obtained by the above screening method by a known method. The epitope of the antibody is prepared by making a synthetic peptide containing the identified epitope or a synthetic peptide mimicking the stereo structure of the epitope. Further, an antibody which binds to an epitope identical to an epitope to which the monoclonal antibody of the present invention binds to human CCR1 is obtained can be obtained by identifying an epitope of an antibody obtained by the above screening method, A part of the synthetic peptide of the identified epitope or a synthetic peptide mimicking the stereo structure of the epitope is produced and immunized. 2. Production of Gene Recombinant Antibody As a production example of a recombinant gene antibody, a method for producing a human type chimeric antibody and a humanized antibody is disclosed below. The recombinant mouse antibody, rat antibody, rabbit antibody, and the like can also be produced by the same method. (1) Gene recombinant antibody expression vector construction Gene recombinant antibody expression vector is an animal cell expression vector in which a DNA encoding CH and CL of a human antibody is incorporated, and DNA encoding CH and CL encoding human antibodies can be used. The cells were separately cultured and constructed using an expression vector. The C region of the human antibody can use CH and CL of any human antibody. For example, a CH of a γ1 subtype of a human antibody, a CL of a kappa type, and the like are used. The cDNA encoding CH and CL of human antibodies uses cDNA, but chromosomal DNA containing exons and introns can also be used. The expression vector for animal cells can be used as long as it can assemble and express a gene encoding a C region of a human antibody. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)] or pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] and the like. As a promoter and promoter in the expression vector for animal cells, an initial promoter of SV40 can be cited [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia virus LTR [Biochem. Biophys. Res Commun., 149, 960 (1987)] or the promoter of the immunoglobulin H chain [Cell, 41, 479 (1985)] and the promoter [Cell, 33, 717 (1983)] and the like. The gene for recombinant antibody expression can be used as an antibody H in terms of the ease of constructing the gene expression vector, the ease of introduction into animal cells, and the balance of the expression levels of the antibody H chain and the L chain in the animal cell. A gene-recombinant antibody expression vector of a type (tandem type) in which a chain and an L chain are present on the same carrier [J. Immunol. Methods, 167, 271 (1994), and an antibody H chain and an L chain may be present on different carriers. Type. As a vector for expressing a gene recombinant antibody in tandem type, pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)] and the like are used. (2) Acquisition of cDNA encoding the V region of an antibody derived from an animal other than human and analysis of an amino acid sequence. Acquisition of cDNA of VH and VL of a non-human antibody and analysis of an amino acid sequence can be carried out as follows get on. cDNA was synthesized by extracting mRNA from fusion tumor cells producing non-human antibodies. The cDNA synthesized is cloned into a vector such as a phage or a plastid to prepare a cDNA gene pool. A recombinant phage or recombinant plastid having a cDNA encoding VH or VL is isolated from the gene pool using DNA encoding a C region portion or a V region portion of a mouse antibody as a probe. The entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plastid is determined, and the full amino acid sequence of VH or VL is estimated based on the base sequence. Animals other than humans which produce non-human antibody-producing fusion tumor cells can be used as mice, rats, hamsters, rabbits, and the like, and any animal capable of producing fusion tumor cells can be used. When whole RNA is prepared from the fusion tumor cells, a kit such as a guanidinium thiocyanate-trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or an RNA easy kit (manufactured by Qiagen) is used. The preparation of mRNA from whole RNA using the oligo(dT) immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], or Oligo-dT30 <Super> mRNA Purification (registered Trademarks) Kits (made by TAKARA BIO), etc. Further, mRNA can be prepared from the fusion tumor cells using a kit such as Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification (registered trademark) Kit (Pharma Pharma). The synthesis of cDNA and the production of the cDNA gene library are performed using a known method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997). ), or a kit such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis (registered trademark) Kit (Stratagene). In the case of producing a cDNA gene bank, a vector which is incorporated into a cDNA synthesized by using mRNA extracted from a fusion tumor cell as a template can be used as long as it can be a vector into which the cDNA can be incorporated. For example, use ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPII (manufactured by Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3-18U (manufactured by Pharmacia), pCD2 [Mol. Cell. Biol., 3, 280 (1983)] or pUC18 [Gene, 33, 103 (1985)] and so on. Escherichia coli which is a cDNA gene library constructed by introducing a phage or a plastid vector can be used as long as it can introduce, express and maintain the cDNA gene pool. For example, XL1-blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)] or JM105 [Gene, 38, 275 (1985)] and the like. A colony/hybridization method using an isotope or fluorescently labeled probe, or a plaque hybridization method using a sample of a VH or VL encoding a non-human antibody from a cDNA gene library [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], etc. Further, a primer is prepared, and a polymerase chain reaction method is performed using a cDNA or cDNA gene library synthesized from mRNA as a template [hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)], whereby cDNAs encoding VH or VL can also be prepared. The selected cDNA is cleaved by a restriction enzyme or the like, and then cloned into a plastid such as pBluescript SK (-) (manufactured by Stratagene), and the nucleotide sequence of the cDNA is determined by a base sequence analysis method or the like which is usually used. The base sequence analysis method is, for example, a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], using ABI PRISM 3700 (manufactured by PE Biosystems) or an ALF DNA nucleic acid sequencer (Pharmacia). A base sequence automatic analysis device, etc., manufactured by the company. The complete amino acid sequence of VH and VL is estimated based on the determined base sequence, and the sequence and protein of the known antibody VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (Septences of Proteins of Immunological Interest, US Dept. Health and Human Services 1991)] A comparison was made to confirm whether the obtained cDNA encodes the complete amino acid sequence of VH and VL of the antibody comprising the secretory signal sequence. The complete amino acid sequence of VH and VL of the antibody comprising the secretory signal sequence, by the sequence and protein of the known antibody VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services ( 1991)] A comparison is made, and the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and the subgroups to which they belong can be known. Further, the amino acid sequence of each of the CDRs of VH and VL can also be obtained by the amino acid sequence of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991) ] Compare and find out. Further, using the obtained complete amino acid sequence of VH and VL, a BLAST method is performed on any database such as SWISS-PROT or PIR-Protein [J. Mol. Biol., 215, 403 (1990)] The source search can confirm whether the complete amino acid sequence of VH and VL is a novel sequence. (3) Construction of human type chimeric antibody expression vector The cDNA encoding the VH or VL of the non-human antibody, respectively, is selected to the CH or CL encoding the human antibody of the gene expression recombinant vector obtained in (1). Upstream of each gene, whereby a human type chimeric antibody expression vector can be constructed. In order to link the 3'-end side of the cDNA encoding the VH or VL of the non-human antibody to the 5'-end side of the CH or CL of the human antibody, a suitable amino acid is encoded by the base sequence of the linking moiety, and A cDNA of VH and VL designed in a manner suitable for restriction of the enzyme recognition sequence. The cDNAs of the produced VH and VL are separately selected in the form of the appropriate expression to the upstream of each gene encoding the human antibody CH or CL of the gene expression recombinant vector obtained in (1). And construct a human type chimeric antibody expression vector. Alternatively, synthetic DNA encoding a non-human antibody VH or VL may be amplified by PCR using synthetic DNA having a recognition sequence of a restriction enzyme at both ends, and the gene recombination obtained in (1) may be selected. A carrier for antibody expression. (4) Construction of cDNA encoding the V region of the humanized antibody The cDNA encoding the VH or VL of the humanized antibody can be constructed as follows. The amino acid sequence of the FR of the VH or VL of the human antibody for the amino acid sequence of the CDR of the VH or VL of the non-human antibody is selected, respectively. The amino acid sequence of the selected FR can be used as long as it is derived from a human antibody. For example, the amino acid sequence of FR of a human antibody registered in a database such as Protein Data Bank, or the consensus amino acid sequence of each subgroup of FR of a human antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and so on. In order to suppress a decrease in the binding activity of the antibody, an amino acid sequence of FR having as much homology as possible (at least 60% or more) of the amino acid sequence of the FR of the original antibody to VH or VL is selected. Next, the amino acid sequence of the CDR of the original antibody is separately grafted into the amino acid sequence of the FR of the VH or VL of the selected human antibody, and the amino acid sequence of the VH or VL of the humanized antibody is designed, respectively. Converting the designed amino acid sequence into a DNA sequence in consideration of the frequency of use of codons seen in the base sequence of the gene of the antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] A DNA sequence encoding the amino acid sequence of VH or VL of a humanized antibody was designed, respectively. Based on the designed DNA sequence, a plurality of synthetic DNAs having a length of about 100 bases are synthesized, and the PCR reaction is carried out using these. In this case, in consideration of the reaction efficiency in the PCR reaction and the length of the DNA which can be synthesized, it is preferred to design six synthetic DNAs for each of VH and VL. Further, by introducing a recognition sequence of a suitable restriction enzyme to the 5' or 3' end of the synthetic DNA located at both ends, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into (1). The gene expression recombinant antibody expression vector. After the PCR reaction, the amplified products were separately cloned into a plastid such as pBluescript SK (-) (manufactured by Stratagene), and the base sequence was determined by the same method as described in (2), and the desired coding was obtained. The plastid of the DNA sequence of the amino acid sequence of the VH or VL of the humanized antibody. Alternatively, the PCR amplification product may be replaced by combining the full-length VH and the full-length VL into one long-chain DNA based on the designed DNA sequence. Further, by introducing a suitable restriction enzyme sequence from both ends of the synthetic long-chain DNA, the cDNA encoding the VH or VL of the humanized antibody can be easily colonized to the expression of the recombinant antibody obtained in (1). Carrier. (5) Alteration of the amino acid sequence of the V region of the humanized antibody. Regarding the humanized antibody, if only the CDRs of the VH and VL of the non-human antibody are grafted to the FR of the VH and VL of the human antibody, the antigen binding activity thereof Reduced compared to the original non-human antibody [BIO/TECHNOLOGY, 9, 266 (1991)]. With respect to humanized antibodies, an amino group directly interacting with an antigen, an amino acid group that interacts with an amino acid of a CDR, and an amino group that interacts with an amino acid of a CDR, is identified by the amino acid sequence of the FR of the human antibody. An acid residue, and an amino acid residue that maintains the steric structure of the antibody and is indirectly related to binding to the antigen, and the amino acid residue is replaced with an amino acid residue of the original non-human antibody, thereby improving Reduced antigen binding activity. In order to identify amino acid residues related to the antigen-binding activity of FR, by using X-ray crystallographic analysis [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 ( 1994)], etc., and the construction and analysis of the stereostructure of the antibody can be performed. Further, several kinds of modifications were made for each antibody, and the correlation between each of them and the antigen-binding activity was examined, and the humanized antibody having the desired antigen-binding activity was obtained by repeated trial and error. The amino acid residues of the FH of the human antibody and the FR of the VL can be modified by performing the PCR reaction described in (4) using the modified DNA. The base sequence was determined by the method described in (2) for the amplification product after the PCR reaction, and it was confirmed that the target modification was carried out. (6) Construction of humanized antibody expression vector The cDNA of the VH or VL of the recombinant antibody encoding the gene is separately selected to the CH or CL encoding the human antibody of the gene expression vector obtained in (1). A humanized antibody expression vector can be constructed upstream of each gene. For example, by introducing the appropriate restriction enzyme at the 5' or 3' end of the synthetic DNA at both ends of the synthetic DNA used in constructing the VH or VL of the humanized antibody obtained in (4) and (5). The sequences are each selected to be upstream of each of the genes encoding the CH or CL of the human antibody of the vector for expression of the recombinant antibody expressed in (1) in such a manner as to be expressed in a suitable form. (7) Temporary expression of the recombinant antibody using the recombinant antibody expression vector obtained in (3) and (6), or the expression vector obtained by the modification, for the temporary expression of the recombinant antibody, and may have The antigen-binding activity of a plurality of human-type chimeric antibodies and humanized antibodies produced was evaluated efficiently. As the host cell into which the expression vector is introduced, any cell can be used as long as it is a host cell capable of expressing the recombinant antibody, for example, COS-7 cells are used [ATCC, American Type Culture Collection number: CRL1651] [Methods] In Nucleic Acids Res., CRC press, 283 (1991)]. The introduction of the expression vector into COS-7 cells is carried out by the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press (1991)], or by lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and so on. After introduction of the expression vector, the expression amount and antigen-binding activity of the recombinant antibody in the culture supernatant are determined by using the enzyme immunoassay method [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual , Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Test Guide, Kodansha Scientific (1987), etc. (8) Stabilization of the expression of the recombinant antibody and the preparation of the recombinant antibody can be obtained by introducing the recombinant antibody expression vector obtained in (3) and (6) into a suitable host cell. A transformant strain that expresses a recombinant antibody. The introduction of the expression vector into the host cell is carried out by electroporation [JP-A-2-257891, Cytotechnology, 3, 133 (1990)]. The host cell into which the recombinant antibody expression vector is introduced can be used as long as it is a host cell capable of expressing the recombinant antibody. For example, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), rat myeloma cell YB2/3HL .P2.G11.16Ag.20 (ATCC number: CRL1662, or YB2/0), mouse myeloma cell NS0, mouse myeloma cell SP2/0-Ag14 (ATCC number: CRL1581), mouse P3X63 -Ag8.653 cells (ATCC No.: CRL1580), Dihydroforate Reductase (hereinafter referred to as dhfr) deficient CHO cells (CHO/DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] and so on. Further, a host cell such as an α1,6-fucosyltransferase gene-deficient CHO cell (International Publication No. 2005/035586, International Publication No. 02/31140), and a lectin-resistant Lec13 can be used. Somatic Cell and Molecular Genetics, 12, 55 (1986)], etc., a protein such as an enzyme related to the synthesis of intracellular sugar nucleotide GDP-fucose in the above host cell, which binds to the α-α of fucose N-glycoside binds to the N-acetyl glucosamine at the reducing end of the complex sugar chain, and the protein related to the sugar chain modification at the 6th position is related to the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus. The activity of the protein or the like is reduced or absent. After introducing the expression vector, the transformant strain stably expressing the recombinant antibody is cultured in a medium for animal cell culture containing a drug such as G418 sulfate (hereinafter referred to as G418), and is selected by the method (Japanese Patent Laid-Open No. Hei 2-257891) Bulletin). The culture medium for animal cell culture is RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL 301 medium (manufactured by JRH Co., Ltd.), IMDM medium (manufactured by Invitrogen), or Hybridoma-SFM medium (manufactured by Invitrogen Co., Ltd.). Or a medium or the like in which various additives such as FBS are added to the medium. The recombinant antibody was expressed in the culture supernatant by culturing the obtained transformant in a medium. The expression amount and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by an ELISA method or the like. In addition, the amount of expression of the recombinant antibody produced by the transformant can be increased by using the dhfr gene amplification system (JP-A No. 2-257891). The recombinant anti-system is purified from the culture supernatant of the transformed strain using a protein A-column [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988) )]. Further, methods such as gel filtration, ion exchange chromatography, and ultrafiltration for protein purification may be combined. The molecular weight of the H chain, L chain or antibody molecule of the purified recombinant antibody can be determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western dot method [Monoclonal Antibodies - Principles and Practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], etc. 3. Evaluation of activity of purified monoclonal antibody or the antibody fragment The purified monoclonal antibody of the present invention or the evaluation of the activity of the antibody fragment can be carried out as follows. The antibody of the present invention or the binding activity of the antibody fragment to human CCR1 was measured using the flow cytometer described in the above 1-(6). Further, it can be measured by a fluorescent antibody method [Cancer Immunol. Immunother., 36, 373 (1993)] or the like. The activity of the antibody of the present invention or the antibody fragment for inhibiting migration of human CCR1 expressing cells by human CCL15 can be measured by the above chemotaxis assay. The CDC activity or ADCC activity of human CCR1 expressing cells can be determined by a known assay [Cancer Immunol. Immunother., 36, 373 (1993); Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan Et al., John Wiley & Sons, Inc., (1993)] performed the assay. 4. Method for controlling effector activity of an antibody As a method for controlling effector activity of a monoclonal antibody of the present invention, fucose which binds to α1,6 to N-acetylglucosamine (GlcNAc) is also known (also known as fucose The method of controlling the amount of core fucose) (International Publication No. 2005/035586, International Publication No. 2002/31140, International Publication No. 00/61739), the above-mentioned N-acetylglucosamine (GlcNAc) exists Method for controlling the reducing end of an N-binding complex type sugar chain bound to aspartic acid (Asn) No. 297 of the Fc region of an antibody; or by modifying an amino acid residue of an Fc region of an antibody Wait. The effector activity of the monoclonal antibodies of the present invention can be controlled by any of the above methods. The effector activity refers to an antibody-dependent activity caused by an Fc region of an antibody, and it is known that ADCC activity, CDC activity, or antibody-dependent phagocytosis of an edible cell based on macrophages or dendritic cells (Antibody-dependent) Phagocytosis, ADP activity), etc. As a method for measuring the activity of the effector, for example, an inflammatory cell as a target, a human peripheral blood mononuclear ball (PBMC) as an effector, and an inflammatory cell-specific antibody can be mixed and cultured for about 4 hours, and then measured. Free lactate dehydrogenase (LDH), an indicator of cytotoxicity. Alternatively, for example, an antibody that recognizes a blood cell-specific antigen such as CD20 can be added to human whole blood, and after the culture, the decrease in the number of labeled blood cells, that is, the effector activity, is measured. Alternatively, for example, other target cells can be mixed in human whole blood, and then specific antibodies can be added to the target cells and cultured, and the decrease in the number of target cells, that is, the effector activity, can be measured. In either case, the effector activity can be freed by free LDH 51 It is measured by a Cr method or a flow cytometry method. The effector activity of the antibody can be increased or decreased by controlling the amount of core fucose of the N-binding complex sugar chain of the Fc of the antibody. As a method for reducing the content of fucose bound to the N-binding complex type sugar chain of the Fc bound to the antibody, an antibody can be obtained by expressing an antibody using CHO cells deficient in the α1,6-fucosyltransferase gene. An antibody that does not incorporate fucose. Antibodies that do not incorporate fucose have higher ADCC activity. On the other hand, as a method of increasing the content of fucose bound to the N-binding complex type sugar chain of the Fc bound to the antibody, the host cell which introduces the α1,6-fucosyltransferase gene is used to express the antibody. Thereby, an antibody that binds to fucose can be obtained. The antibody that binds to fucose has ADCC activity lower than that of the antibody that does not bind fucose. Further, ADCC activity or CDC activity can be increased or decreased by modifying the amino acid residue of the Fc region of the antibody. The CDC activity of the antibody can be increased, for example, by using the amino acid sequence of the Fc region described in the specification of U.S. Patent Application Publication No. 2007/0148165. Further, the ADCC activity or the CDC activity can be increased or decreased by modifying the amino acid described in the specification of U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775, or U.S. Patent No. 7,317,091. Further, the antibody of the present invention also includes an antibody having a half-life controlled in blood, which is carried out by an amino acid modification or a sugar chain modification corresponding to the constant region of the above antibody, for example, Japanese Patent Laid-Open No. 2013- The amino acid described in Japanese Patent Laid-Open No. 165716 or Japanese Patent Laid-Open No. 2012-021004, etc., is modified to control the reactivity to the Fc receptor, thereby controlling the half-life in blood. Further, by using the above method in combination with one type of antibody, an antibody having an effector activity or a half-life of blood can be obtained. 5. The method for treating a disease using the anti-human CCR1 monoclonal antibody or the antibody fragment of the present invention The monoclonal antibody or the antibody fragment of the present invention can be used for treating a CCR1-related disease as long as it is a human CCR1-dependent cell migration, a lesion, or the like Any human CCR1-related disease. The therapeutic agent containing the monoclonal antibody or the antibody fragment of the present invention may be one containing only the antibody or the antibody fragment as an active ingredient, but usually mixed with one or more pharmacologically acceptable carriers. It is provided in the form of a pharmaceutical preparation manufactured by a method known in the art of pharmacy. Examples of the administration route include oral administration, or oral administration such as intraoral, intratracheal, intrarectal, subcutaneous, intramuscular or intravenous. Examples of the administration form include a spray, a capsule, a tablet, a powder, a granule, a syrup, an emulsion, a suppository, an injection, an ointment, a patch, and the like. Examples of the preparation suitable for oral administration include emulsions, syrups, capsules, troches, powders, granules, and the like. For example, a liquid preparation of an emulsion or a syrup may be a sugar such as water, sucrose, sorbitol or fructose, a glycol such as polyethylene glycol or propylene glycol, an oil such as sesame oil, olive oil or soybean oil, or a paraben. A preservative such as a preservative or a fragrance such as strawberry flavor or peppermint is produced as an additive. For capsules, troches, powders or granules, excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, and polyvinyl alcohol are used. A binder such as hydroxypropyl cellulose or gelatin, a surfactant such as a fatty acid ester, or a plasticizer such as glycerin or the like is produced as an additive. As a preparation suitable for parenteral administration, there are an injection, a suppository, a spray, and the like. The injection is produced using a carrier containing a salt solution, a glucose solution or a mixture of the two, or the like. The suppository is produced using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. The spray is produced by using a carrier which does not cause irritation to the oral cavity and the tracheal mucosa of the recipient, and which allows the monoclonal antibody or the antibody fragment of the present invention to be dispersed as fine particles to be easily absorbed. As the carrier, for example, lactose or glycerin or the like is used. Alternatively, it can be produced in the form of an aerosol or a dry powder. Further, a component exemplified as an additive exemplified for a preparation suitable for oral administration may be added to the above-mentioned parenteral preparation. 6. A method for diagnosing a disease using the anti-human CCR1 monoclonal antibody or the antibody fragment of the present invention, using the monoclonal antibody or the antibody fragment of the present invention, detecting or measuring human CCR1 or human CCR1 expression cells, thereby diagnosing human CCR1 Related diseases. The diagnosis of a cancer, an autoimmune disease, and an inflammatory disease of a human CCR1-related disease can be performed, for example, by detecting or measuring human CCR1 present in a patient by immunological methods. Further, diagnosis can be performed by detecting human CCR1 expressed in cells in a patient by immunological methods such as flow cytometry. The immunological method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or an antibody. For example, a radioactive substance labeling immunological antibody method, an enzyme immunoassay, a fluorescent immunoassay, a luminescent immunoassay, a Western blotting method, or a physicochemical method are used. The radioactive substance-labeled immunological antibody system, for example, reacts an antigen or an antigen-expressing cell with the antibody of the present invention or the antibody fragment, and further reacts with a radiolabeled anti-immunoglobulin antibody or the antibody fragment, and then uses a scintillation counter or the like. The measurement was carried out. In the enzyme immunoassay, for example, an antigen or an antigen-presenting cell is reacted with the antibody or the antibody fragment of the present invention, and further reacted with an anti-immunoglobulin antibody or a binding fragment labeled with an enzyme or the like, and then the substrate is added to absorb the light. The absorbance of the reaction solution was measured with a photometer. For example, a sandwich ELISA method or the like is used. As the label used in the enzyme immunoassay, an enzyme label of a known [Enzyme Immunoassay, Medical College (1987)] can be used. For example, an alkaline phosphatase label, a peroxidase label, a luciferase label, or a biotin label is used. The sandwich ELISA method is a method in which an antibody is bound to a solid phase, and an antigen as a detection or measurement target is captured, and the captured antigen is allowed to react with the second antibody. In the ELISA method, two kinds of antibodies or antibody fragments which recognize the antigen to be detected or measured are prepared, and the antigen recognition sites are different, and the first antibody or the antibody fragment is previously adsorbed to the well plate (for example, a 96-well plate). Then, the second antibody or the antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. The well plate to which the above antibody is adsorbed is reacted with the cell isolated from the living body or the disrupted liquid, the tissue or the disrupted solution thereof, the cell culture supernatant, the serum, the pleural effusion, the ascites or the eye liquid, and the labeled The monoclonal antibody or the antibody fragment is reacted to carry out a detection reaction corresponding to the labeled substance. The antigen concentration in the test sample is calculated based on a calibration curve prepared by phase-diluting the antigen with known concentration. As the antibody used in the sandwich ELISA method, any of a plurality of antibodies or monoclonal antibodies can be used, and Fab, Fab' or F(ab) can also be used. 2 And other antibody fragments. The combination of the two antibodies used in the sandwich ELISA method may be a combination of a single antibody or an antibody fragment that recognizes a different epitope, or a combination of a plurality of antibodies and a monoclonal antibody or an antibody fragment. Fluorescence immunoassay is carried out by the method described in the literature [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Monoclonal Antibody Test Guide, Kodansha Scientific (1987)]. As the label used in the fluorescent immunoassay, a fluorescent label known as [Fluorescent Antibody Method, Soft Science (1983)] can be used. For example, use FITC or RITC. The luminescent immunoassay is measured by the method described in the literature [Bioluminescence and Chemiluminescence Clinical Examination 42, Hirokawa Shusho (1998)] and the like. Examples of the label used in the luminescent immunoassay include a known illuminant label, and acridinium ester or azole. Western blotting method uses SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gelatin) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)] to retain antigen or antigen expressing cells, etc., The gel is blotted to a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane, and the membrane is reacted with an antibody or antibody fragment recognizing the antigen, and further labeled with a fluorescent substance such as FITC and passed through an oxidase. After the reaction is carried out by an enzyme label or an anti-mouse IgG antibody or a binding fragment such as biotin label, the label is visualized and thereby measured. Hereinafter, an example will be disclosed. A cell or tissue having a polypeptide having the amino acid sequence of SEQ ID NO: 2 is dissolved, and electrophoresis is carried out by SDS-PAGE in a number of 0.1 to 30 μg of protein per lane under reducing conditions. The electrophoresed protein was transferred to a PVDF membrane, and subjected to a blocking operation by reacting with PBS containing 1 to 10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes. Here, it is reacted with the monoclonal antibody of the present invention, washed with PBS containing 0.05 to 0.1% of Tween-20 (hereinafter referred to as Tween-PBS), and treated with oxidase-labeled goat anti-mouse IgG. The reaction was carried out for 2 hours under temperature. The polypeptide having the amino acid sequence of SEQ ID NO: 2 was detected by washing with Tween-PBS and detecting the band in which the monoclonal antibody was bound using ECL Western Blotting Detection Reagents (manufactured by Amersham). As an antibody used for detection by the Western blotting method, an antibody capable of binding to a polypeptide which does not maintain a natural stereostructure is used. The physicochemical method is carried out, for example, by forming an aggregate by binding human CCR1 as an antigen to the monoclonal antibody of the present invention or the antibody fragment, and detecting the aggregate. In addition, as a physicochemical method, a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Clinical Examination Method, Jinyuan Publishing (1998)] may be used. In the latex immunoturbidimetric method, if a carrier such as polystyrene latex having a particle size of 0.1 to 1 μm sensitized by an antibody or an antigen is used, and an antigen-antibody reaction is caused by a corresponding antigen or antibody, the scattered light in the reaction solution is used. Increase, through light reduction. The change as the absorbance or the turbidity of the integrating sphere is detected, thereby measuring the antigen concentration and the like in the test sample. A well-known immunological assay can be used for the detection or measurement of human CCR1 expression cells. Among them, immunoprecipitation, immunocytochemistry, immunohistochemical staining, or fluorescent antibody staining are preferably used. In the immunoprecipitation method, a human CCR1 expression cell or the like is reacted with the monoclonal antibody or the antibody fragment of the present invention, and then a carrier having specific binding energy to an immunoglobulin such as a protein G-Sepharose or the like is added to cause an antigen-antibody complex. precipitation. Alternatively, it can also be carried out by the following method. The above-described monoclonal antibody or the antibody fragment of the present invention was subjected to solid phase formation to a 96-well plate for ELISA, and then blocked with BSA-PBS. When the antibody is in an unpurified state such as a culture supernatant of a fusion tumor, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G, etc. are previously immobilized to ELISA. The 96-well plate was blocked with BSA-PBS, and the supernatant of the fusion tumor culture was dispensed to bind. Then, BSA-PBS was removed and washed thoroughly with PBS, and then reacted with a solution of human CCR1 expressing cells or tissues. The immunoprecipitate was extracted from the well-washed well plate by SDS-PAGE using a sample buffer, and detected by the above Western blot method. The immunocyte staining method or the immunohistochemical staining method is a method in which an antigen-expressing cell or tissue is treated with a surfactant, methanol, or the like in order to optimize the passage of the antibody, and the individual is treated with the present invention. The antibody reaction is further reacted with an anti-immunoglobulin antibody or a binding fragment such as a fluorescent label such as FITC, an enzyme such as an oxidase or a biotin label, or the like, and the label is visualized and observed under a microscope. Further, it can be detected by fluorescent antibody staining [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), monoclonal antibody experimental guide, Kodansha Scientific (1987)], that is, fluorescently labeled antibody After reaction with the cells, the solution was analyzed by flow cytometry. In particular, the monoclonal antibody or the antibody fragment of the present invention which binds to human CCR1 can detect cells which retain the natural stereostructure by fluorescent antibody staining. In the fluorescent antibody staining method, when the FMAT8100HTS system (Applied Biosystems, Inc.) or the like is used, it is not necessary to form the antibody-antigen complex and the free antibody or antigen which are not involved in the formation of the antibody-antigen complex. The amount of antigen or the amount of antibody can be determined by performing isolation. Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples. [Examples] [Example 1] Production of human and mouse CCR1 expression vectors (1) Preparation of each CCR1 gene The CCR1 or CCR1-CCR3 chimeric receptors of human or mouse encoding the following 1 to 7 were synthesized. DNA (GenScript Japan). The restriction enzyme sites (BamHI and NotI) and the Kozak sequence for incorporation into each vector were added during the synthesis. 1. A cDNA sequence encoding human CCR1 (hereinafter referred to as hCCR1) (SEQ ID NO: 1) 2. A cDNA sequence encoding mouse CCR1 (hereinafter referred to as mCCR1) (SEQ ID NO: 3) 3. Coding human CCR3 (hereinafter referred to as hCCR3) cDNA sequence (SEQ ID NO: 5) 4. Chimeric receptor encoding the amino acid sequence of human CCR1 No. 1 to No. 31 substituted with the corresponding N-terminal amino acid sequence of human CCR3 (hereinafter referred to as The cDNA sequence of NC3-hCCR1) (SEQ ID NO: 6) 5. Encoding the amino acid sequence of No. 1 to No. 31 of mouse CCR1 to the corresponding N-terminal amino acid sequence of human CCR3. The cDNA sequence of the receptor (hereinafter referred to as NC3-mCCR1) (SEQ ID NO: 7) 6. Encoding the amino acid sequence No. 171 to No. 194 of human CCR3 to amino acid No. 171 to No. 194 of human CCR1 The cDNA sequence of the chimeric receptor derived from the base acid sequence (hereinafter referred to as hCCR3_EL2hCCR1) (SEQ ID NO: 8) 7. The substitution of the amino acid sequence No. 171 to No. 194 of human CCR3 to the 171th of mouse CCR1 The cDNA sequence of the chimeric receptor (hereinafter referred to as hCCR3_EL2mCCR1) obtained from the amino acid sequence No. 194 (hereinafter referred to as hCCR3_EL2mCCR1) (2) Preparation of human CCR1 expression vector Restriction enzymes BamHI and NotI (New England Biolab Co.) treated DNA (1) -1 synthesized above encoding the hCCR1, the DNA fragment was purified. The Tol2 transposon vector (International Publication No. 2010/143698) (hereinafter referred to as Tn-pMug-Hygro) was treated with the same restriction enzyme, mixed with a DNA fragment encoding CCR1, and then treated by DNA ligase (TAKARA BIO). Make it link. The ligated DNA was introduced into E. coli competent cells (TAKARA BIO Co., Ltd.), and an Escherichia coli strain having the target plastid DNA was selected from colonies having obtained drug resistance. This Escherichia coli strain was cultured again, and DNA for transfection was purified from the culture solution (hereinafter, the plastid thus produced is referred to as hCCR1/Tn-pMug-Hygro). (3) Preparation of various CCR expression vectors The mCCR1, hCCR3, NC3-hCCR1, NC3-mCCR1, hCCR3_EL2hCCR1, hCCR3_EL2mCCR1 and Tn-pMug-Hygro synthesized in the above (1) are linked by the same method as (2) above. , constructing expression vectors (hereinafter referred to as mCCR1/Tn-pMug-Hygro, hCCR3/Tn-pMug-Hygro, NC3-hCCR1/Tn-pMug-Hygro, NC3-mCCR1/Tn-pMug-Hygro, hCCR3_EL2hCCR1/Tn-pMug, respectively -Hygro, hCCR3_EL2mCCR1/Tn-pMug-Hygro). (4) Preparation of mCCR1 expression vector The DNA encoding mCCR1 synthesized in the above (1) and the pair pCAGGS [Gene. 1991 Dec 15; 108(2): 193- were prepared by the same method as (2) above. 9.] Attach the internal ribosomal entry site (IRES, internal ribosomal entry site) and the vector obtained by the neomycin resistance gene, pCAG-IRES-neo, to construct a performance vector (hereinafter referred to as mCCR1/pCAG-IRES-neo). . [Example 2] Production of CCR1 expression cell line (1) Preparation of hCCR1 expression cell The hCCR1/Tn-pMug-Hygro and Tol2 transposase expression vector TPEX_pMug (International Publication No. 2013) prepared as the plastid DNA produced in Example 1 was prepared. /005649) was introduced into CHO-S (Thermo Fisher Scientific) to produce a expressing cell strain. The gene introduction was carried out in the following manner using Fugene HD (Promega Corporation). Will be prepared to 1 × 10 5 The cells of cell/mL were seeded to a 6-well plate at 2.5 mL per well, and after 24 hours, a mixture of hCCR1/Tn-pMug-Hygro, TPEX_pMug, and Fugene HD was added to the culture solution. After 72 hours from the addition, 1 mg/mL of hygromycin (Invitrogen) was added, and the drug selection was carried out for about 2 weeks. The cells which obtained the drug tolerance were collected, and the expression analysis was performed by a flow cytometer (FACS Calibur, BD Biosciences), and the expression of the introduced hCCR1 was confirmed. This cell line was called CHO-S-hCCR1. (2) Preparation of various CCR-expressing cells mCCR1/Tn-pMug-Hygro, hCCR3/Tn-pMug-Hygro, NC3-hCCR1/Tn-pMug-Hygro, NC3-mCCR1/Tn-pMug prepared in Example 1 -Hygro, hCCR3_EL2hCCR1/Tn-pMug-Hygro, and hCCR3_EL2mCCR1/Tn-pMug-Hygro were introduced into CHO-S cells by the same method as (1) above to prepare a expressing cell strain. Hereinafter, the cell lines are referred to as CHO-S-mCCR1, CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, CHO-S-hCCR3_EL2hCCR1 and CHO-S-hCCR3_EL2mCCR1, respectively. (3) Preparation of RL33-hCCR1 cells The hCCR1/Tn-pMug-Hygro produced in Example 1 and the Tol2 transposase expression vector TPEX_pMug (International Publication No. 2013/005649) were introduced into a rabbit cell line RL-33 [Yoshii] Et al., Jpn J Med Sci Biol. 1977 Jun; 30(3): 149-57], producing hCCR1 expressing cell lines. The gene introduction was carried out in the following manner using Lipofectamine LTX (Thermo Fisher Scientific). Will be prepared to 1 × 10 5 Cells of cell/mL were seeded to 6-well plates at 2 mL per well, and a mixture of 2.5 μg of plastid DNA and 5 μL of Lipofectamine LTX was added to the medium. After 1 hour of the addition, 1 mg/mL of hygromycin was added, and the drug selection was carried out for about 2 weeks. The cells obtained by the drug tolerance were recovered, and the expression analysis was performed by a flow cytometer, and the expression of the introduced hCCR1 was confirmed. Hereinafter, this cell strain is referred to as RL33-hCCR1. (4) Preparation of RL33-mCCR1 cells mCCR1/pCAG-IRES-neo produced in Example 1 (4) was introduced into RL-33 by the same method as (3) above to prepare a expressing cell strain. The drug selection was carried out using 0.5 mg/mL of G418. Hereinafter, this cell strain is referred to as RL33-mCCR1. [Example 3] Preparation of anti-CCR1 rabbit polyclonal antibody An anti-CCR1 rabbit polyclonal antibody was produced by the following method. The N-terminal peptide of human CCR1 (SEQ ID NO: 10) was synthesized, and two rabbits (New Zealand White) were immunized once every two weeks for a total of five times. Complete Freund's Adjuvant (CFA) was used only for the first immunization, and Incomplete Freund's Adjuvant (IFA) was used after the second immunization, and subcutaneous injection was performed at several sites on the back. After immunization, serum was collected from individuals having an increased antibody titer, and affinity purification was performed using a Protein A column (GE Healthcare) to purify IgG. The anti-CCR1 rabbit polyclonal antibody thus produced was referred to as E5971. [Example 4] Expression analysis by flow cytometry (1) Confirmation of CCR1 expression The CCR1 expression cell strain produced in Example 2 was stained using the anti-CCR1 rabbit polyclonal antibody E5971 produced in Example 3, and used. Flow cytometry (FCM) confirmed the performance of CCR1. The FCM analysis is performed as follows. Put the cells at 2×10 5 The cell/well was seeded into a 96-well plate and washed with a staining buffer [3% FBS (Thermo Fisher Scientific) / DPBS (Nacalai Tesque) / 0.1% sodium azide (Nacalai Tesque)]. The cells were treated with 10 μg/mL of E5971 on ice for 1 hour, washed with staining buffer, and then added with secondary antibody Alexa Fluor 647 goat Anti-Rabbit IgG at a final concentration of 1 μg/mL (Thermo Fisher Scientific Made by the company), treated at room temperature for 30 minutes. The cells were washed again with a staining buffer, suspended in a staining buffer, and analyzed using BD FACSCalibur (BD Biosciences). From this, it was confirmed that the introduced CCR1 was expressed in the produced CCR1 expressing cell line. (2) Confirmation of CCR3 performance The performance of CCR3 was also confirmed by the same method as (1) above for the CHO-S-hCCR3 produced in Example 2. The primary anti-system used commercially available 444-11 antibody (MBL) as an anti-CCR3 antibody, and the secondary anti-system used Alexa Fluor 647 goat Anti-mouse IgG (H+L) (Thermo Fisher Scientific). From this, it was confirmed that the introduced CCR3 was expressed in CHO-S-hCCR3. [Example 5] Production of monoclonal antibody using CCR1 knockout mouse In order to obtain mouse cross-over antibody, commercially available CCR1 knockout (KO) mouse (B6.129S4-Ccr1) was used. tm1Gao N10+N5) (Non-Patent Document 17) (Taconic Co., Ltd.) was used to prepare a monoclonal antibody. The antibody production was carried out as follows. (1) Immunosensitization CHO-S-hCCR1, CHO-S-mCCR1, RL33-hCCR1 and RL33-mCCR1 produced in Example 2 were used as immunogens. Use 1×10 for each immunization 7 Cells / only. For CCR1 KO mice 5 to 9 weeks old, Alum gel (LSL) (80 μL/mouse) and pertussis vaccine (Nacalai Tesque) (1×10) were added only for the first immunization. 7 The cells/only) were used as adjuvants for immunosensitization by intraperitoneal administration. Each immunization was prepared by using PBS in such a manner that the dose was 500 μL/mouse. The second immunization was performed 2 weeks after the initial immunization, and the third immunization was performed one week later, and partial blood collection was performed 3 days later. (2) Antiserum evaluation (FCM) Using various CCR1 expression cells prepared in Example 2, specific antibody titers in serum were determined by FCM. The measurement was carried out in accordance with the following procedure. Using 1% BSA (Nacalai Tesque)-PBS (Nacalai Tesque) [containing 0.02% EDTA (Nacalai Tesque), 0.05% NaN 3 (Nacalai Tesque)] prepared cells into 1×10 5 Cell/well was dispensed at 50 μL/well to the bottom of a 96-well cell culture plate. Among them, the serum collected from the immunized animal as the sample to be tested was treated with 1% BSA-PBS (0.02% EDTA, 0.05% NaN) at 50 μL/well. 3 ) Prepared at a final concentration of 200-fold dilution, 1000-fold dilution, and 5000-fold dilution, and placed at 4 ° C for 30 minutes. After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated and the particles of the cells were dispersed using a plate shaker. Dispense 1% BSA-PBS (0.02% EDTA, 0.05% NaN) at 200 μL/well 3 After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated, and the particles of the cells were dispersed by a plate shaker. 1% BSA-PBS (0.02% EDTA, 0.05% NaN) was dispensed thereto at 50 μL/well. 3 ) Alexa Fluor 647, goat anti-mouse IgG (H+L) or Alexa Fluor 488 goat anti-mouse IgG (H+L), which was finally 300-fold diluted, was placed under light-shielding at 4 ° C for 30 minutes. After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated and the particles of the cells were dispersed using a plate shaker. Dispense 1% BSA-PBS (0.02% EDTA, 0.05% NaN) at 200 μL/well 3 After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated, and the particles of the cells were dispersed by a plate shaker. 1% BSA-PBS (0.02% EDTA, 0.05% NaN) was dispensed at 50 μL/well 3 The fluorescence intensity was measured by a flow cytometer [FACSCanto (trademark) II/BD]. Thereby, the individual who confirmed the enhancement of the antibody was selected and the spleen was taken. (3) Preparation of fusion tumor by cell fusion Mouse myeloma cell line P3-U1 (P3X63Ag8U.1, ATCC CRL-1597) was cultured by S-Clone cloning medium (Eidia), and was used as serum-free adaptation. The parent strain of cell fusion. The spleen of the immunized animal was extracted under aseptic conditions, and hemolyzed by red blood cell lysis buffer (RED BLOOD CELL LYSING BUFFER) (Sigma-Aldrich), and then washed twice with PBS to spleen cells with P3-U1. The number of cells was mixed as spleen cells: P3-U1 = 8:1, and centrifuged (1200 rpm, 5 minutes). After fully dispersing the cell population of the obtained precipitated portion, 1 g of polyethylene glycol-1000 (PEG-1000, Pure Chemical Co., Ltd.) and 1 mL of MEM medium (Nacalai Tesque Co., Ltd.) were added at 37 ° C while stirring. And 0.5 mL of a mixture of 0.35 mL of dimethyl hydrazine (Sigma-Aldrich), 1 mL of MEM medium was added every 1 minute, and after 5 times, MEM medium was added in a total amount of 50 mL. . The cell suspension was centrifuged (900 rpm, 5 minutes), and the cells of the obtained precipitated fraction were slowly dispersed, and the spleen cells were 1.5×10. 7 The cell concentration of cell/9 mL was suspended in S-Clone cloning medium to which HAT SUPPLEMENT (Thermo Fisher Scientific) was added. The cloning medium to which HAT was added was previously dispensed at 100 μL/well into a 96-well culture plate, and the cell suspension was dispensed at 100 μL/well in CO. 2 Incubator (5% CO 2 Incubate for 8 to 10 days at 37 ° C). (4) Screening of fusion tumor The binding activity to CCR1 of the antibody contained in the culture supernatant of the fusion tumor was evaluated by FCM. The test sample was a fusion tumor culture supernatant, and the staining and measurement were carried out by the same procedure as in the above (2). (5) Sub-selection of the fusion tumor The cells in the wells which are positive in the screening are sub-selected and cultured in the culture medium for about 7 to 10 days. (6) Determination of antibody subtypes Subtypes of each antibody were determined by using FCM of a subtype-specific secondary antibody. The dyeing and measurement were carried out by the same procedure as in the above (2). The test sample was cultured using a fusion tumor. The detection system used Alexa Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific), and each subtype specific antibody (Alexa Fluor 488 goat anti-mouse IgG1 (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse IgG2a (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse IgG2b (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse IgG3) (Thermo Fisher Scientific). (7) The antibody from the supernatant of the fusion tumor culture supernatant was purified from the culture supernatant of the fusion tumor selected as described above. For the purification, Protein G Sepharose 4 Fast Flow (GE Healthcare) was used. The culture supernatant was centrifuged to remove precipitates, which were filtered using a filter. The column was filled with 400 μL of the carrier, and the buffer was replaced with DPBS. The culture supernatant was added, and after the antibody was adsorbed to the monomer, it was washed twice with 10 mL of DPBS. After dissolving by adding 0.4 mL of IgG Elution Buffer (Thermo Fisher Scientific), it was neutralized with 0.1 mL of 1 M Tris-HCl (NIPPON GENE) pH 8.6. Desalting was performed using a NAP column (GE Healthcare) and the buffer and DPBS were replaced for subsequent analysis. The strain names, sources and subtypes of the produced antibodies are shown in Table 1. [Table 1] [Example 6] THP-1 migration (chemotaxis) analysis As a human cell strain expressing CCR1, a human mononuclear leukemia cell line THP-1 was known. This cell is known to exhibit chemotaxis corresponding to a concentration gradient of a CCR1 ligand such as CCL3, CCL5, CCL15 or CCL23, and a migration assay using THP-1 is a general CCR1 inhibitor evaluation system. Therefore, for the anti-human CCR1 antibody obtained in Example 5, the experimental system was also used to evaluate whether or not the activation of human CCR1 caused by human CCL15 was inhibited. The method of migration analysis is described below. THP-1 cells were obtained from ATCC. THP-1 cells were cultured in the presence of 5 μM All-trans-retinoic acid (ATRA, Wako Pure Chemical Industries, Ltd.) for 3 days, recovered after differentiation induction, and heated to 37 ° C. The assay medium [1% FBS (Thermo Fisher Scientific) / RPMI 1640 (Nacalai Tesque)] was washed and then suspended in the same medium. Prepared as 1×10 6 Cell/mL, cells were dispensed at 100 μL/well to a top layer of Transwell (Corning, #3421) with a pore size of 5 μm. The assay medium supplemented with 1 ng/mL recombinant human CCL15 (68 aa) (R&D technologies, #628-LK) was injected as a chemical attractant in the lower layer at 37 ° C in 5% CO. 2 After 4-6 hours of incubation in the incubator, the number of cells moved to the lower layer was quantified using Celltiter-Glo (Promega). When the cell migration of the purified antibody was evaluated using the present assay system, 90 μL of the cell suspension was mixed with 10 μL of the purified antibody solution in a 1.5 mL test tube, and cultured at 37 ° C for 1 hour, and then the cells were dispensed to Trasnwell. Upper layer. The anti-system was used for the measurement after adjusting the final concentration to 0.3, 1, 3, and 10 μg/mL. The results obtained are shown in Fig. 1 (a) and Fig. 1 (b). As shown in Fig. 1 (a) and Fig. 1 (b), mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody obtained in Example 5 The KM5955 antibody and the KM5956 antibody all inhibited the migration of THP-1 induced by activated CCL15 in a concentration-dependent manner. From the above, it is understood that the mouse anti-human CCR1 monoclonal antibody of the present invention is an antibody which inhibits the activation of human CCR1 caused by human CCL15. [Example 7] Determination of human CCR1 binding region of anti-human CCR1 antibody Human CCR1 was expressed using CCR1-CCR3 chimeric receptor and measuring mouse anti-human CCR1 monoclonal antibody obtained in Example 5 by FCM Combine the area. The measurement was carried out by the same method as in Example 4. As CCR1-CCR3 chimeric receptor-expressing cells, CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, and CHO-S-hCCR3_EL2hCCR1 produced in Example 2 were used. Also, CHO-S was used as a negative control. As the antibody to be tested, each of the fusion tumor culture supernatant diluted 10-fold, the existing mouse anti-human CCR1 monoclonal antibody 53504 antibody (R&D Technologies), and the mouse anti-human CCR3 monoclonal antibody 444-11 antibody ( MBL company). Regarding the measurement results, the fluorescence intensity when a certain cell is stained with a test antibody (each fusion culture supernatant, 53504 antibody or 444-11 antibody) and a secondary antibody is divided by the cell to be stained only by the secondary antibody. Fluorescence intensity. When the obtained value is 10 or more, it is judged that the test antibody binds to the cell, and when it is less than 10, it is judged that the test antibody is not bound to the cell, and is shown as ○ and × in Table 2, respectively. [Table 2] According to Table 2, mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody were not bound to CHO-S-hCCR3, but combined At CHO-S-hCCR3_EL2hCCR1. Therefore, it is understood that the mouse anti-human CCR1 monoclonal antibody of the present invention binds to the extracellular loop 2 of human CCR1. [Example 8] Chemotactic analysis of existing anti-human CCR1 antibody and anti-human CCR1 antibody (1) Preparation of existing mouse anti-human CCR1 monoclonal antibody 2D4 antibody Obtained from ATCC to produce an existing anti-human CCR1 antibody That is, a fusion tumor of the 2D4 antibody (U.S. Patent No. 6,756,035) (LS-125-2D4-11-10-1). The fusion tumor was cultured using Hybridoma-SFM (Thermo Fisher Scientific), and the antibody was purified from the culture supernatant. For the purification, Protein G Sepharose 4 Fast Flow (GE Healthcare) was used. The culture supernatant was centrifuged, and the obtained culture supernatant was filtered through a filter. The column was filled with 400 μL of the carrier, and the buffer was replaced with DPBS. The culture supernatant was added to the column, and the antibody was adsorbed to the carrier, and then washed twice with 10 mL of DPBS. After 0.4 mL of IgG Elution Buffer (Thermo Scientific) was added to the column to elute the antibody, 0.1 mL of 1 M Tris-Cl (Nacalai Tesque) pH 8.6 was used to neutralize the antibody solution. The antibody solution was desalted using a NAP column (GE Healthcare) and the buffer and DPBS were replaced for subsequent analysis. The purified 2D4 antibody was subjected to SDS-PAGE under reducing conditions by a usual method, and it was confirmed that the antibody was purified. Further, the binding activity of the 2D4 antibody to human CCR1 was confirmed by the method described in Example 4 and by FCM. The 2D4 anti-system was reacted at 0.1, 1 μg/mL, and for the cells, CHO-S-hCCR1 was used as a human CCR1 expression cell, and CHO-S was used as a negative control. As a result, the 2D4 antibody did not bind to CHO-S, but bound to CHO-S-hCCR1 in a concentration-dependent manner. From this, it was confirmed that the purified 2D4 antibody has binding to human CCR1 in the same manner as the commercially available 2D4 antibody. (2) Chemotaxis analysis The anti-human CCR1 antibody and the mouse anti-human CCR1 antibody monoclonal antibody KM5908 antibody and KM5916 antibody obtained in Example 5 were assayed for inhibition of CCR1 according to the method described in Example 6. The activity of activation was compared between the results obtained for each antibody. As the conventional anti-human CCR1 antibody, 2D4 antibody, 141-2 antibody (MBL company) and 53504 antibody (R&D Systems, Inc.) produced in (1) were used. The results obtained are shown in Fig. 2. As shown in Fig. 2, 2D4 antibody, 141-2 antibody and 53504 antibody, which are conventional anti-human CCR1 antibodies, did not inhibit the migration of THP-1 cells induced by activated CCL15, and on the other hand, mouse anti-human CCR1 single The strain antibody KM5908 antibody and KM5916 antibody inhibited the migration of the above cells in a concentration-dependent manner. As described in Example 6, the anti-human CCR1 antibody obtained in Example 5 inhibited the migration of THP-1 cells induced by activated CCL15 in a concentration-dependent manner under the same experimental conditions as in the present example [Fig. 1 (Fig. 1) a) and Figure 1 (b)]. Therefore, the existing anti-human CCR1 antibody did not inhibit the activation of human CCR1 caused by human CCL15, and on the other hand, the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 obtained in Example 5. Antibodies, KM5915 antibodies, KM5916 antibodies, KM5954 antibodies, KM5955 antibodies, and KM5956 antibodies are all antibodies that inhibit the activation of human CCR1 by human CCL15. [Example 9] Production of recombinant antibody (1) Colonization and sequence determination of antibody variable region gene Total RNA was extracted from the fusion tumor selected in Example 5 using Trizol (Life Technologies), by 5' The -RACE method allows amplification of the antibody gene. The RACE cDNA synthesis system uses the SMARTer RACE Kit (Clontech). The antibody variable region is obtained by PCR using a primer having specificity for a sequence to be added during cDNA synthesis in RACE, and a primer for amplification of mouse Ig γ chain or kappa chain (SEQ ID NO: 11-14) The fragment was amplified, subjected to colonization, and the base sequence of the DNA fragment was confirmed. With respect to each of the anti-human CCR1 antibodies obtained in Example 5, the base sequence of the amino acid sequence encoding the variable regions of the heavy and light chains, the amino acid sequence presumed based on the base sequence, and The sequence numbers of the amino acid sequences obtained by removing the signal sequence of the amino acid sequence are shown in Table 3, respectively. Further, the sequence numbers of the amino acid sequences indicating the CDRs of the respective antibodies of the present invention are shown in Table 4, respectively. [table 3] [Table 4] (2) Preparation of Expression Vector of Chimeric Antibody The constant region in each of the anti-human CCR1 antibodies produced in Example 5 was replaced with an amino acid modified containing S228P, L235E and R409K by the method described below. A chimeric antibody derived from the human IgG4 constant region (human IgG4PE_R409K). The plastid DNA of the nucleotide sequence encoding the amino acid sequence of the variable region of each antibody produced in (1) was used as a template, and PCR was carried out by using a primer having the same base sequence for recombination. Base sequence amplification of the amino acid sequence encoding the variable region of each antibody. Using the In-Fusion HD Cloning Kit (Clontech), the base sequence was replaced with the N5KG4PE R409K vector [the N5KG1 vector (U.S. Patent No. 6,001,358), the base sequence encoding the constant region of human IgG1 was replaced with the coding described above. A vector (hereinafter referred to as N5KG4PE R409K vector) obtained by modifying the nucleotide sequence of the constant region of human IgG4 modified by amino acid is linked to produce a expression vector of a chimeric antibody. The experimental procedure is based on the guidelines that accompany the kit. (3) Production and purification of chimeric antibody A chimeric antibody was produced using the expression vector produced in (2) and the Expi293 Expression System (Life Technologies). The steps are performed in the following manner in accordance with the accompanying guide. Expi293F cells (Thermo Fisher Scientific) at 2×10 6 The cell/mL density was incubated at 37 ° C for 24 hours, followed by 1.25 × 10 per reaction. 8 The cell was added to 42.5 mL of Expi293 Expression Medium (Thermo Fisher Scientific). 50 μg of plastid DNA and ExpiFectamin 293 Reagent (Thermo Fisher Scientific) were added to Opti-MEM (Thermo Fisher Scientific), and after allowing to stand for 30 minutes, the plastid solution was added to the above cell solution. After further culturing, ExpiFectamin 293 Transfection Enhancer (culture amount totaling 50 mL) was added to the cell liquid. After the cell solution was cultured for 7 to 10 days, the culture supernatant was collected. For the purification of the antibody, Protein G Sepharose 4 Fast Flow (GE Healthcare) was used. The recovered culture supernatant was centrifuged, and the obtained culture supernatant was filtered through a filter. The column was filled with 400 μL of the carrier, and the buffer was replaced with DPBS. The culture supernatant was added to the column, and after the antibody was adsorbed to the monomer, the column was washed twice with 10 mL of DPBS. After 0.4 mL of IgG Elution Buffer (Thermo Scientific) was added to the column to elute the antibody, 0.1 mL of 1 M Tris-Cl pH 8.6 was added to the antibody solution for neutralization. The antibody solution was desalted using a NAP column (GE Healthcare) for subsequent resolution. The chimeric antibodies of the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody thus obtained are described as chKM5907 antibody, chKM5908, respectively. The antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody. [Example 10] Evaluation of binding ability of chimeric antibody The chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody, and chKM5956 antibody produced in Example 9 were evaluated. According to the method described in Example 4, the binding to human and mouse CCR1 was measured by FCM. As human CCR1 expression cells and mouse CCR1 expression cells, CHO-S-hCCR1 and CHO-S-mCCR1 produced in Example 2 were used, respectively. The results show that the chKM5955 antibody binds to human CCR1. It was also shown that other chimeric antibodies bind to human and mouse CCR1. [Example 11] Chemotaxis analysis using chimeric antibody The chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 prepared in Example 9 were used. The antibody was assayed for activity against inhibition of human CCR1 activation according to the method described in Example 6. The results showed that each chimeric antibody inhibited the migration of THP-1 caused by activation of human CCL15. The present invention has been described in detail with reference to the specific embodiments of the invention, and various modifications and changes can be made without departing from the spirit and scope of the invention. In addition, this application is based on a Japanese patent application filed on January 20, 2016 (Japanese Patent Application No. 2016-8983), the entire contents of which are incorporated herein by reference.